The prognostic significance of lymphocytic infiltration in malignant melanoma of the choroid.

Lymphocytic and plasmocytic infiltration surrounding a malignant tumor probably represents an immunologic response of the host directed against the neoplasm. It has been documented that lymphocytic infiltration has a favorable effect on prognosis in a number of human tumor systems, including cutaneous melanoma. In the present study, 309 consecutive cases of choroidal melanoma were examined for cellular infiltration, and these data were correlated with other histologic parameters and with prognosis. A intense cellular infiltration was found in 15 tumors (4.9%) and a moderate infiltration in 37 (12.0%). Tumors with cellular infiltration were significantly larger and more vascularized than the remaining tumors, which may be related to the accessibility of the immune system to the tumor. There was a higher percentage of poorly differentiated tumors among the tumors with cellular infiltration. However, it does not appear the cellular infiltration favorably influences the prognosis of choroidal melanoma. The 5-year survival rates of patients with like cell types did not depend upon the degree of cellular infiltration demonstrated by the primary tumor. Although choroidal melanomas are capable of inciting an immune response, this response is apparently ineffective in preventing metastatic spread.


Introductory statement on the progress of The National Large Bowel Cancer Project: its goals, objectives, and programs.

The introductory lecture updates the state of the art bearing on cancer of the large bowel. Topics discussed include etiological factors, the pathology of the disease, the role of animal models, the early diagnosis of the disease, including biochemical markers, with emphasis on immunology, and finally therapy, including surgery, radiotherapy, chemotherapy, and the immunological approaches to therapy as identified in the ongoing activities of the National Large Bowel Cancer Project. Interesting aspects of complementing the traditional ways of treating colorectal disease are noted in the discussion of new approaches to chemotherapeutic treatment based on rational developments obtained throught the study of cell kinetics and cell turnover. It is pointed out that this and other interrelationships considered in the multidisciplinary experimental and clinical approaches provide important leads toward the understanding of the mechanisms of disease, discovery of causes and means of prevention, methods of earlier diagnosis, improved rational modalities of treatment, and, one hopes, more successful management.


The large bowel carcinogenic effects of hydrazines and related compounds occurring in nature and in the environment.

Five substituted hydrazines that induce large bowel and other types of cancer in laboratory animals are described. Two of these compounds, which originate in nature, are 1,1-dimethylhydrazine, a tobacco ingredient, and methylhydrazine, formed from a chemical present in the edible wild mushroom Gyromitra esculenta. The human population is therefore exposed to them considerably. In addition, both compounds are manufactured and used in rocket fuel. The other three chemicals, 1,2-dimethylhydrazine dihydrochloride,1-methyl-2-butylhydrazine dihydrochloride and trimethylhydrazine hydrochloride, are manufactured synthetically only and apparently are not found in substantial quantities in the environment.


Nitrosamines and nitrosamides in the etiology of gastrointestinal cancer.

It has been amply demonstrated that N-nitroso compounds are formed by interaction of nitrite with secondary and tertiary amino compounds, and that this reaction can occur in vivo as well as in food processed with nitrite. The favored site of reaction in vivo is the stomach. It is likely that N-nitroso compounds formed in this way, especially nitrosamides, could reach the colon and contribute to the induction of colon tumors.


Inhibition of carcinogenic effect of bracken fern (Pteridium aquilinum) by various chemicals.

The inhibitory effect of butylated hydroxyanosole (BHA), disulfiram, calcium chloride, and polyvinylpyrrolidone (PVP) on the intestinal and urothelial carcinogenicity of bracken fern (BF) was determined in albino rats. Of 10 groups of rats, one group received a normal diet, one received a BF-containing diet (one-third of diet by weight), four received a normal diet with one of the following supplements, and four received BF-containing diet with one of the following: BHA (5 mg/g diet); disulfiram (5 mg/g diet); PVP (50 mg/g diet); or calcium chloride (20 mg/g diet). At 12 months of the experiment, the following  results were noted: in the BF-treated group, 30 rats (100%) exhibited intestinal tumors and 22 of 30 (73%) urinary bladder tumors. In the BF-BHA group, 15 of 20 rats (75%) showed intestinal tumors and 12 of 20 rats (60%) urinary bladder tumors. Of the 16 rats in the BF-disulfiram group, 12 (80%) had intestinal and 10 (62.5%) had urinary bladder tumors. In the BF-calcium chloride group, intestinal tumors arose in 16 of 23 rats (70%) and urinary bladder tumors in 4 of 23 rats (17%), while in the 28 BF-PVP rats, 26 (93%) exhibited tumors of the intestine and 5 (18%) tumors of the urinary bladder. Dietary BHA, disulfiram and calcium chloride decreased the incidence of intestinal tumors by about 25--30% (p less than 0.01). Similarly, PVP and calcium chloride inhibited BF-induced urinary bladder carcinogenesis by about 80% (p less than 0.001). No tumors were detected in groups receiving either normal diet or normal diet supplemented with BHA, disulfiram, calcium chloride or PVP.


Biology of colon cancer: an overview.

The altered growth characteristics of neoplastic cells have recently been associated with changes in membrane glycoproteins present on the cell surface. Since the carbohydrate moieties of surface membrane glycoproteins are asymmetrically located on the external cell surface, these glycoconjugates are likely candidates for providing cell surfaces with many of their biological properties. Using specific external cell surface labeling techniques, we have broadened our investigation of tumor cell surface glycoconjugates to include studies on cultured human epithelial cells from fetal intestine and from colonic carcinoma. We have isolated by affinity chromatography and gel filtration and integral membrane glycoprotein, termed Galactoprotein I, from a cultured human colonic adenocarcinoma cell line, which appears to be identical in many respects to CEA. Further examination of cell surface glycoproteins regarding quantitative and qualitative alterations and topographical redistribution should provide an insight into the biological aspects of tumor development.


Studies on translocation of immunoglobulins across intestinal epithelium.

The issue of how immunoglobulins are transported across the intestinal epithelium into the gut lumen was examined by immunohistocytochemical techniques. IgA, IgM, IgG and secretory component (SC) in human small intestine at light and electron microscopic levels were localized by the peroxidase-labeled antibody technique. By light microscopy, IgA, IgM and SC, but not IgG, were found associated with columnar epithelial cells in gland crypts. By electron microscopy, SC was found in the perinuclear membranes, rough endoplasmic reticulum and Golgi complexes of these cells. IgA and IgM as well as SC were found in the basolateral plasma membranes and cytoplasmic vacuoles of the cells. It was concluded that in the human small intestine 1) SC is synthesized by columnar secretory epithelial cells; 2) IgA and IgM, but not IgG, are transported through these cells; 3) IgA and IgM could combine during transport with SC on plasma membranes or within the cytoplasm of these cells. In additional experiments, in vitro binding of peroxidase-labeled dimeric IgA to specific sites, corresponding to the sites of SC, on human intestinal epithelial cells, was demonstrated.


Influence of gonadal hormones and age on 1,2-dimethylhydrazine-induced colon carcinogenesis.

BD-II and BD-IX male and female rats received weekly subcutaneous (s.c.) injections of 15 mg/kg 1,2-dimethylhydrazine dihydrochloride (DMH) beginning at either 35, 120 or 210 days of age and continuing for 20 weeks. Control animals received only the DMH vehicle. Additional BD-II and BD-IX male and female rats of the three age groups were gonadectomized at 21, 106 and 196 days. Beginning 14 days after gonadectomy, the rats received 15 mg/kg of DMH by s.c. injection once a week for 20 weeks. Animals were sacrificed 35 weeks after the initial DMH injection. Control rats of the appropriate age and sex did not develop colon tumors. BD-IX rats are apparently more sensitive to DMH than BD-II rats. The incidence of DMH-induced cancer is less in females than in males in both the BD-II and BD-IX animals. Gonadectomy does not affect cancer incidence in either BD-II males or females nor in the BD-IX females but reduced the incidence in BD-IX males exposed initially at either 120 or 210 days. Administration of androgen to castrate BD-IX males (120-day-old group) increases the incidence of colon cancer to that approaching the intact animal but has little effect in the BD-II castrate male. These data suggest a genetically influenced susceptibility to DMH-induced colon carcinogenesis between BD-II and BD-IX rats. Furthermore, a sex difference is evident in both BD lines but age appears to be a factor only in older BD-IX females. Apparently, androgens influence DMH-induced tumorigenesis in BD-IX males only if the initial exposure of DMH occurs after sexual maturity.


Ultrastructure of the "transitional" mucosa adjacent to large bowel carcinoma.

The transitional mucosa immediately adjacent to large bowel carcinoma was examined in 13 patients by scanning electron microscopy (SEM), and this was correlated with transmission electron microscopy (TEM) and light microscopy (LM). Marked abnormalities were present in this mucosa in 9/13 patients with distortion and loss of the normal architecture. Examination of adenomas showed abnormalities also to be present adjacent to the neoplastic mucosa. In all patients, the transitional mucosa was compared with two pieces of normal mucosa; in one patient, typical changes of transitional mucosa were found in this region. In the remaining patients, normal mucosa by SEM was found to consist to large deep furrows (primary crypts) as well as the crypts of Lieberkuhn (secondary crypts). The secondary crypts structure showed marked individual variation between patients but one type was present in nine patients. It is uncertain whether this is a pattern with a high incidence in the general population, or whether patients with this type of mucosa are particularly predisposed to large bowel carcinoma. SEM may, therefore, play a part in identifying patients particularly likely to develop large bowel carcinoma.


Phenotypic markers in human skin fibroblasts as possible diagnostic indices of hereditary adenomatosis of the colon and rectum.

Hereditary adenomatosis of the colon and rectum (ACR) and its Gardner's syndrome variant, an autosomal dominant trait, indicate a propensity for neoplasia. The present study describes the growth abnormalities of cultured human skin fibroblasts derived from normal-appearing cutaneous biopsies of ACR genotypes and a portion of the clinically asymptomatic ACR progeny, first filial generation, and their differential susceptibility to transformation by Kirsten murine sarcoma virus. These skin fibroblasts, but not cells derived from unaffected individuals, showed lack of contact inhibition, decreased serum requirement for growth, elevated levels of plasminogen activator, and alterations in the intracellular distribution of actin cables; they did not, however, grow in the absence of anchorage, nor did they form palpable tumors in congenitally athymic BALB/c nu/nu mice, and they were normal with regard to cholesterol feedback regulation. Skin fibroblasts from ACR subjects were 100- to 1000-fold more susceptible to transformation by the Kirsten murine sarcoma virus than were normal cells. The virally transformed skin fibroblasts were anchorage-independent and formed tumors in athymic mice. These growth abnormalities represent steps in the changing phenotypic expression of cells undergoing neoplastic transformation. Identification of abnormal expressions associated with oncogenesis may facilitate their use as diagnostic indices for the detection of latent forms of colon cancer in man.


Surgical and medical measures in prevention of large bowel cancer.

Traditional methods for prevention of large bowel cancer rely on surveillance of patients with known precursors of bowel cancer, namely ulcerative colitis and those genetically linked polyposis syndromes that have malignant potential. Identification of heritable bowel cancer families and solitary polyp--cancer families provide additional populations that merit intensive scrutiny. Persuasive, if circumstantial, evidence suggests that maintaining patients free of large bowel polyps reduces the risk of developing large bowel cancer. Prospects for prevention of large bowel cancer are extended by recognition that a diet low in fat may reduce the risk of large bowel cancer. Furthermore, there is considerable evidence in animals that a variety of antioxidants limit large bowel carcinogenesis and preliminary evidence in man that these agents may control large bowel neoplasia.


Methylation of intestinal and hepatic DNA in rats treated with methylazoxymethanol acetate.

Descending colon is the most sensitive segment of rat intestine and is at least as sensitive as liver to the carcinogenic effects of methylazoxymethanol acetate. To determine whether a relationship exists between tumor induction and level of DNA methylation, we measured the levels of 7-methylguanine in the DNA isolated from duodenum, descending colon, and liver of rats treated with this carcinogen. Because radiolabeled methylazoxymethanol acetate is not available, we utilized high pressure liquid chromatography whereby methylated purines could be detected in amounts as little as 100-300 pmoles. DNA isolated from liver of carcinogen-treated rats had significant amounts of 7-methylguanine. On the contrary, DNA isolated from descending colon of rats treated with methylazoxymethanol acetate had minimal amounts of 7-methylguanine; these data suggest that the level of 7-methylguanine does not correlate with sensitivity to tumor induction by methylazoxymethanol acetate.


Changes in chromosomal proteins in colon cancer: the complexity and DNA-binding properties of tumor-associated proteins and evidence for their association with the malignant state in human colonic epithelium.

The properties of two classes of nonhistone nuclear proteins (NHNP) whose appearance has been correlated with the process of carcinogenesis in the colonic epithelium of rats given 1,2-dimethylhydrazine (DMH) have been investigated. NHNP isolated from adenocarcinomas of the colon were tested for their binding to homologous DNA. The tumor-specific protein class TNP1 (molecular weight ca. 44,000) shows high affinity for DNA while the second class of tumor-specific proteins (TNP2; molecular weight ca. 62,000) does not bind to DNA. When tumor chromatin is subjected to a limited digestion with DNAse I under conditions that are known to preferentially digest active genes, the TNP1 proteins are selectively released, suggesting that this protein fraction is associated with the actively transcribing portions of the genome. The complexity of both tumor-specific protein classes has also been analyzed by two-dimensional gel electrophoresis. This method reveals a high degree of complexity both in TNP1 and TNP2. Protein classes similar to TNP1 and TNP2 that are also found in human colonic adenocarcinomas are not detectable in polyps from familial polyposis-affected patients at times when no sign of malignancy has yet appeared.


Feasibility of fecal occult-blood testing for detection of colorectal neoplasia: debits and credits.

A screening program for colorectal cancer and adenomas has been applied to 6,579 mostly asymptomatic men and women age 40 years and older utilizing fecal occult-blood testing followed by investigation of patients with positive slides by air-contrast barium enema and colonoscopy. A control population of 7,325 patients had sigmoidoscopy only and no occult-blood testing. Approximately 1% of the patients had positive slides; most patients had only one or two slides positive. Approximately 50% of patients with positive slides had significant neoplastic lesions, including 23 patients with large adenomas and 7 patients with cancers. Pathological staging of cancers was more favorable in the screened asymptomatic group as compared with the control group. Neoplastic lesions seen on sigmoidoscopy in screened patients who had negative fecal occult-blood tests included 2 cancers and 15 large adenomas. Reasons for false negativity include possible conversion of initially positive slides to negative. Screening for colorectal cancer and adenomas with fecal occult-blood testing appears to be feasible approach with good patient compliance, and manageable rate of positive slides productive of a high percentage of neoplastic lesions. The number of false-positives seems to be low. False negativity has been observed and will require further study.


Hereditary polyposis coli I: the diagnostic value of colonoscopy, barium enema, and fecal occult blood.

In families with one of the Mendelian hereditary polyposes, one can predict the proportion of patients at risk and thus obtain a denominator against which colonoscopy, barium enema, and fecal occult blood can be validated. Colonoscopy is more sensitive than barium enema. There were 42 positive colonoscopies, 12 positive barium enemas, both being positive in 10 of these. There were 141 negative enemas, 133 negative colonscopies, and 118 negative for both. Occult blood was positive in 30% of patients with polyposis, five to seven times more frequently than in those without evident polyposis. Colonoscopy detected polyposis in 30% of the progeny of affected people. The shortfall, compared with the 50% expected under the Mendelian hypothesis, is readily explained by removal of affected cases from the study by surgery or death from cancer.


Selective inhibition with sodium cyanate of protein synthesis in colon cancer cells.

Sodium cyanate, which in its tautomeric acidic form, isocyanic acid, acts as a protein carbamylating reagent, has been previously shown to inhibit selectively both DNA and protein synthesis in a variety of solid tumors. We have now compared its effects on protein synthesis in normal colonic epithelium and in colon tumors induced by the administration of 1,2-dimethylhydrazine to rats. The incorporation of 3H-amino acids into cytoplasmic and nuclear protein fractions was suppressed to a much greater extent in the tumor tissue than in colonic epithelial tissue surrounding the tumors of cyanate-treated rats. Despite its effect on tumor protein synthesis in whole animals, cyanate had little or no effect on cultured cells (HT-29) derived from a human adenocarcinoma of the colon, nor on other malignant cell lines such as HeLa S3 cells, chick fibroblasts transformed by the Rous sarcoma virus, mouse Ehrlich ascites tumor cells, or rat Novikoff hepatoma cells. However, the administration of cyanate i.p. does suppress amino acid incorporation by Novikoff hepatoma cells in the peritoneal cavity of rats. The implication that the mechanism of cyanate inhibition of protein synthesis in tumors may require its in vivo metabolism or utilization to produce a postsynthetic modification of circulatory factors is discussed.


Restoration of in vitro growth control to malignant cells.

A glycoprotein (molecular weight, ca. 160,000) from culture medium of contact-inhibited hamster melanocytes restores contact inhibition of growth to malignant melanocytes of man, mouse, and hamsters, and also effectively inhibits growth in vitro of a broad spectrum of malignant and normal cell types of ectodermal, mesodermal and endodermal origins, including human colon carcinomas. The melanocyte contact inhibitory factor (MCIF) produces G1 growth arrest in malignant melanocytes; inhibition of all cell types is reversible, does related, and nontoxic at concentrations below 200 microgram/ml, but selectively lethal to malignant cells at higher concentrations. An electrophoretically identical protein is present in culture media of contact-inhibited melanocytes, fibroblasts, and epidermal cells, but absent from those of colon carcinomas, HeLa cells and malignant melanomas. Nevertheless, an MCIF-like band is present in whole cell homogenates of human colon carcinomas and hamster melanomas. MCIF may permit normal surface interactions required for feedback inhibition of growth.


Human colonic tumor cell kinetics: potential for therapy.

Using new in vitro techniques developed at the Cancer Research Unit, cell kinetic measurements were obtained in primary and metastatic human colonic tumors, polyps and normal bowel that did not require in vivo 3HTdR and required only single samples of tissue. These techniques included the measurement of the number of cells in DNA synthesis (LI), an estimate of the DNA synthesis time (Ts) and the growth fraction of tissues by means of the primer-available DNA-dependent DNA polymerase assay (PDP). From these data, the potential doubling time and the cell cycle time (Tc) of the tumors were calculated. Early preliminary data on human colonic specimens presented in Tables 1 and 2 indicate that there is an increase in LI from the low polyps to higher adenocarcinomas. There is little difference between primary and metastatic tumor cell kinetics. Growth fraction estimates (PDP) of the various colonic tissue types are also not significantly different and except for villous adenomas, DNA synthesis times are constant. The median 3HTdR labeling indices of 7% primary adenocarcinomas include a number of samples (approximately 20% of all samples) with high labeling indices (in the 10--20% range). These high labeling tumors may be those that show objective response to S-phase active drugs, e.g., 5-FU.


Adjuvant immunotherapy and chemoimmunotherapy in colorectal cancer (Dukes' class C): prolongation of disease-free interval and survival.

The poor postsurgical prognosis in patients with colorectal cancer of the Dukes' C classification has prompted a clinical trial of adjuvant immunotherapy versus chemoimmunotherapy intended to prolong either the disease-free interval or the overall survival or both. One hundred and twenty-one patients have been entered on this study. Fifty-two patients received BCG alone and 69 patients received the combination of 5-FU and BCG. The disease-free interval and the overall survival were compared with similar parameters in a group of historical controls with similar prognostic characteristics who were operated on in our institution prior to the initiation of the current study. There was no difference as yet between BCG alone and the combination of 5-FU + BCG in terms of both the disease-free interval and the survival. Both treatments, however, had significantly better results than the surgical controls. Adjuvant therapy, especially with BCG is advocated for patients with colorectal carcinoma, Dukes' C class, following potentially curative surgery.


Active specific immunotherapy potential for the treatment of large bowel cancer.

Active specific immunotherapy, harnessing the strength and specificity of the host immune response to destroy neoplastic cells, may offer an ideal surgical adjuvant treatment modality for human colon cancer. Unfortunately, achievement of this goal has been obscured by 1) the effect of excess residual disease to interfere with the host's destructive response, 2) the weak nature of tumor resistance, 3) the potential adverse effect of concomitant treatments such as chemotherapy, and 4) the present limitation of poorly defined immunogens to induce, as well as insensitive assay systems to detect, host sensitizaion. Recent immunologic and chemical research revealing distinctive surface membrane structures on colon cancer cells suggests that a controlled trial of irradiated, autochtonous cell vaccines (without mycobacterial adjuvants) may provide a new therapeutic tool for Dukes B2 and C stages of human colon cancer.


Tissue changes resulting from the injection of gamma-irradiated cells into the gynogenetic teleost, Poecilia formosa.

In previous studies a clone of the gynogenetic fish, Poecilia formosa (the Amazon molly), was successfully used as a test animal to identify ultraviolet-induced damage. This work has been extended, and the fish system was used to detect damage caused byionizing radiation. Fish cells, exposed in vitro to 250 and 500 rads, were injected into young isogenic recipients, and 9 months later the fish were examined grossly and histologically. Two of the most conspicuous changes that resulted were the development of extensive invasive thyroid hyperplasia and hypertrophy body, with an apparent reduction in the amount of hemato-poietic tissue in the head kidney and spleen. We discuss the difference between the responses of the recipient fish to cells exposed to ionizing radiation and to cells exposed to ultraviolet light.


Separation of cells from mouse solid tumors by centrifugal elutriation.

Centrifugal elutriation was used to separate cells dissociated from two hypotetraploid mouse solid tumors, a fibrosarcoma and a sarcoma derived from L-P59 cells, based on their sedimentation rates. The separation was rapid, requiring less than 1 hr; yielded about 80 percent cell recovery; and resulted in little loss of cell viability. Aanlysis of DNA content by flow cytometry demonstrated the synchrony obtained with these tumor cells. The fractions with the lowest sedimentation rates contained predominantly normal cells, those with intermediate sedimentation rates contained predominantly tumor cells in the G1 phase of the cell cycle, and those with the highest sedimentation rates contained mostly tumor cells in S or G2. The clonogenicity of L-P59 cells, assayed in culture, markedly increased with increasing sedimentation rates. In contrast, the clonogencity of fibrosarcoma cells, assayed in vivo by a lung colony assay, was lower for the smaller cells, but was essentially constant among the larger cells. Autoradiography of cells labeled in vivo with tritiated thymidine demonstrated no differences in the proportions of cycling cells in various fractions. These results demonstrate that subpopulations differing in cell type, phase of the cell cycle, and clonogencity can be rapidly separated from solid tumors by centrifugal elutriation.


Rosette formation and inhibition in cervical dysplasia and carcinoma in situ.

Total and early rosettes and rosette inhibition were measured in patients with cervical dysplasia and carcinoma in situ. Both total and early rosettes were significantly depressed in patients with carcinoma in situ; early rosettes were also significantly lower than were controls in women with severe dysplasia. Rosette inhibition titers were increased in most patients with moderate dysplasia and in all patients with either severe dysplasia or carcinoma in situ. Thus, the Rosette inhivition test may be useful in detecting, in a precancerous state, patients at risk for cancer.


Correlation of cytotoxicity and mitotic spindle dissolution by vinblastine in mammalian cells.

In two hamster cell lines that differed 100-fold in their vinblastine sensitivity, dissolution of the mitotic spindle by vinblastine in living cells correlated with cytotoxicity from vinblastine expressed as suppression of colony formation. The effect on the spindle apparatus occurred in 30 sec or less and thus provides a rapid assay for determining the cytotoxic effects of the Vinca alkaloids, as well as the potential for quantitative assay of solutions of Vinca derivatives.


Morphogenesis of early 1, 2-dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacter freundii.

The morphogenesis of 1, 2-dimethylhydrazine (DMH)-induced lesions in the colon of outbred NIH Swiss mice was determined for up to 5 months of treatment. The effect of hyperplasia on DMH carcinogenesis was also evaluated by introducing a transient hyperplastic stimulus to the colon during the chronic weekly treatment regimen of DMH. The hyperplastic stimulus was a naturally occurring disease of mice, transmissible murine colonic hyperplasia, which is caused by a variant of Citrobacter freundii. In control mice, those not receiving the bacterium, weekly injections of the carcinogen induced neoplastic changes first detectable at two months of treatment in all segments of the colon and in both sexes. The changes increased in frequency and severity with time. Diffuse mucosal hyperpladia and chronic inflammatory and degenerative changes were also associated with DMH after prolonged treatment. The hyperplastic stimulus of C. freundii reduced the latent period for appearance of early DMH tumors, but it had no influence on already established DMH tumors.


Kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth.

The kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth were studied. The steady deceleration of the specific growth rate with increasing tumor mass that was observed was attributable to a prolongation of the cell cycle, particularly of the S and G2 phases. The cell cycle time (Tc) was 43.3 hr (TG1 equals 10.8, TS equals 26.8, and TG2 equals 5.7 hr) on the seventh day of growth and 76.0 hr (TG1 equals 14.0, TS equals 52.0, and TG2 equals 10.0 hr) on the tenth day of growth. The growth fraction showed a decrease from 0.77 to 0.60 during the 7- to 10-day tumor growth interval. The cell death rate remained low and essentially unchanged during this period. A high correlation was found between polyamine synthesis (ornithine decarboxylase activity) and the specific growth rate; the correlation coefficient was 0.985. There was also a high positive correlation between the cellular polyamine (spermidine and spermine) and nucleic acid content (spermidine: DNA equals 0.916, spermine: DNA equals 0.947, spermidine:RNA equals 0.907, and spermine: RNA equals 0.881). These observations suggest that there may be a functional coupling between polyamines and nucleic acids, and they support the hypothesis that polyamines play an important role in DNA replication and cell division.


Cellular attachment to implanted foreign bodies in relation to tumorigenesis.

Previous experiments demonstrating a reduction of tumorigenicity by roughening the surfaces of plastics implanted in rodents, or by increasing the pore size of cellulose filter implants, were repeated with observations on cellular attachment to these objects and to filters strengthened and made impermeable by bonding to plastic. Round 13-mm discs of methylmethacrylate implanted S.C. in A/BiF/F50 + mice produced sarcomas in 12 percent of mice at 64 weeks. Tumor incidence increased to 60 percent (p less than 0.001) in mice receiving discs to which cellulose filters with pore sizes of 0.025 to 0.1 micrometer were bonded. No tumors occurred with discs covered by 0.45-micrometer filters, followed up to 83 weeks. Vinyl coverslips 15 mm square also produced no sarcomas when covered by 0.45 micrometer filters; plain vinyl produced sarcomas in 40 percent of mice at 64 weeks (p less than 0.001). Sanding of vinyl surfaces reduced tumorigenicity (p less than 0.05). Permeability, fragility, and storage capacity of filters are apparently not related to tumorigenicity. Surface roughness probably is related. Cells, mostly macrophages, were densely and uniformly attached to nontumorigenic surfaces from 24 hr to 2 years after inplantation but were distinctly fewer and not uniformly distrubuted on tumorigenic surfaces. Topology favoring attachment was inherent in 0.45-mum filters and was produced in plastic by gouging irregular excavations 10 to 15 micrometer deep.


Correlation of DNA distribution abnormalities with cytogenetic findings in human adult leukemia and lymphoma.

Pulse cytophotometric analysis of bone marrow cells from 175 patients with leukemia or lymphoma showed abnormalities of cellular DNA distribution in 29 patients for an overall incidence of 16.6 percent. Comparative standard cytogenetic examination indicated that high-degree chromosomal aberrations (less than or equal to 44, greater than or equal to 53 chromosomes) can generally be detected on DNA histograms, whereas patients with diploid, pseudodiploid, 45-hypodiploid, and 47-hyperdiploid abnormalities usually escape recognition by this technique. There were 11 patients with normal diploid or near-diploid karyotypes exhibiting marked DNA deviations; this discrepancy may reflect lack of proliferation of some leukemic clones which is a prerequisite for cytogenetic identification.


Antitumor effect of two oral steroids, mepitiostane and fluoxymesterone, on a pregnancy-dependent mouse mammary tumor (TPDMT-4).

Pregnancy-dependent TPDMT-4 mammary tumors, characterized by requiring estrogen, progesterone, and pituitary hormones for growth, grew continuously in female DDD mice carrying pituitary isografts. The experimental model was used to investigate the antitumor effects of two p.o. steroids, mepitiostane and fluoxymesterone. When tumors implanted with pituitary isografts into the fat-pad reached palpable size, animals received 6 doses/week of 0.1, 0.3, 1.0, and 3.0 mg of either steroid intragastrically. Mepitiostane significantly suppressed tumor growth with regression in 25 and 29 percent of animals at 1.0 and 3.0 mg, respectively, but had no inhibitory effects at other doses. Fluoxymesterone retarted tumor growth during the first week of treatment at 3.0 mg but finally had no inhibitory effects at any doses. Under similar conditions ovariectomy caused tumor regression immediately, and epitiostanol, the parent steroid of mepitiostane, significantly suppressed tumor growth when given in 3 injections/week of 0.5 mg s.c. Tumous had papillary structures and almost lacked secretory activity. A comparison of these findings to those obtained with earlier generations suggests that TPDMT-4 tumors became less sensitive to the antitumor effect of epitiostanol and were able to grow at lower hormone levels with succeeding generations. Morphologically, progression to more cancer and less secretory activity was noticed.


Enhanced induction of immune resistance by concanavalin A-bound L1210 vaccine and an immunopotentiator prepared from Coriolus versicolor.

Combined administration of a vaccine consisting of a small number (2 X 10(6)) of L1210 murine leukemic cells treated with glutaraldehyde and concanavalin A and a protein-bound polysaccharide preparation of Coriolus versicolor induced synergistic resistance to L1210 leukemia in BALB/c X DBA/2CrF1 mice. This effect was dependent on the dose and timing of the administration of the protein-bound polysaccharide preparation, being most effective at the time of or 1 day after the second vaccination. Induced resistance was not cross-reactive with P388 murine leukemia, indicating specificity of resistance. This immunopotentiation by the protein-bound polysaccharide did not occur when L1210 cells treated with glutaraldehyde, but not with concanavalin A, were used as a vaccine.


Natural cytotoxic reactivity of rat lymphocytes against gross virus-induced tumor cell lines as measured by [125I]iododeoxyuridine and tritiated proline microcytotoxicity assays.

Mononuclear cells from rat spleens that mediate "natural" cytotoxicity are described, and the subpopulation responsible for this activity is partially identified. These naturally cytotoxic cells have been designated "N-cells" and appear to be lymphocytes that lack both detectable immunoglobulin and complement receptors and are therefore not mature B-cells. They are different from the classic "null cells", which have immunoglobulin and/or complement recepters. This subpopulation of cells is responsible for the natural activity present is normal spleens and in spleens from immunized animals. These results indicate that the same subpopulation of cells responsible for natural activity in the short-term assays is also responsible for natural activity in the long-term microcytotoxicity assays reported here. In addition, these mononuclear cells are active against both nonadherent cells and monolayer targets. Natural activity as measured by the long-term assays appears to be somewhat less age specific than that reported previously with the short-term chromium assay. Appropriate base lines are described in an attempt to better document natural activity in these assays. This natural activity must be closely monitored in any system purporting to measure cell-mediated cytotoxicity.


Relationship between effects on nucleic acid synthesis in cell cultures and cytotoxicity of 4-demethoxy derivatives of daunorubicin and adriamycin.

Four new derivatives of daunorubicin and two new derivatives of Adriamycin characterized by the absence of the methoxyl groups at the C-4 position have been studied in cell cultures in vitro to establish structure-activity relationships. 4-Demethoxydaunorubicin was 27 to 100 times more active than was daunorubicin when inhibiting the cloning efficiency of exponential-phase HeLa cells and, like daunorubicin, was slightly active on early plateau-phase cells. DNA synthesis in mouse embryo fibroblasts stimulated by fetal calf serum was inhibited equally by the two compounds, although 4-Demethoxydaunorubicin was slightly more active than was daunorubicin when inhibiting RNA synthesis. The beta anomer of 4-demethoxydaunorubicin showed a reduced activity on HeLa cells compared to its alpha anomer, but it was equally active on DNA synthesis. The stereoisomers of 4-demethoxydaunorubicin bearing the inverted configuration in positions 7 and 9 were devoid of significant cytotoxic activity and were only slightly active on DNA synthesis at the doses tested. 4-demethoxyadriamycin and 4-demethoxy-4'-epi-adriamycin were 65 to 500 times more active than was Adriamycin on HeLa cell cloning efficiency and about 10 times more active on DNA synthesis in mouse embryo fibroblasts. Cell uptake in mouse embryo fibroblasts was also investigated for all the new derivatives tested.


Age-related differences in the trends of fatal Hodgkin's disease as a consequence of immune experience.

It has been proposed by MacMahon, on the basis of the variations among populations in the age distribution of fatal Hodgkin's disease, that there are two etiological factors, one important in young adults and the other important in middle-aged and elderly people. Correa has extended this by suggesting that those who died of the disease as young adults in developed countries would have succumbed to the disease as children under less favorable circumstances. In England and Wales, the mortality rate from Hodgkin's disease doubled from 1911 to 1970. If there were two etiologies, it seems unlikely that they would remain in step with one another over such a long period, when it was clear that some factor of major etiological importance was changing. Examination of the trends with time of the age-specific rates confirmed the previously reported decline in children and rise in elderly people and revealed a rapid rise in the mortality of young adults of both sexes. These changes were not compatible with any reasonable variations in the diagnosis or classification of Hodgkin's disease. Our data are compatible with the hypotheses that the etiology of the disease in young adults in different from that in elderly people and that the rise in the mortality of young adults is due to a transfer of deaths from the disease in childhood.


Stimulation of the synthesis of mouse epidermal histones by tumor-promoting agents.

Topical application of 17 nmoles of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, resulted in a stimulation of the incorporation of [(3)H]lysine into epidermal histones. Maximum incorporation occurred 24 hr after treatment, concurrent with maximum DNA synthesis. The effects of phorbol and two phorbol esters on histone synthesis were related to their tumor-promoting activities. Treatment with hydroxyurea partially prevented the phorbol ester-induced stimulation of both DNA and histone synthesis, although it had no effect on the stimulation of protein synthesis. These findings are consistent with the likelihood that phorbol ester-induced epidermal histone synthesis is the result of a coupling between DNA synthesis and histone synthesis.


Sequential alteration of the pancreas during carcinogenesis in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine.

A systematic histological-pancreatographic study indicated that, during pancreatic carcinogenesis by N-nitrosobis(2-oxopropyl)amine, the ductular cells (cells of intercalated or intralobular ductules and especially those of peri-and intrainsular ductules) are most responsive. The initial proliferation (multiplication) and distension of ductules seemed due to primary hyperplasia of ductular cells, followed by metaplasia, atypia, and malignant alteration. Among 75 induced adenocarcinomas, most were of ductular origin, whereas only a few seemed to arise from ductal epithelium (interlobular, secondary, and main ducts). There was no preferential segment for tumor development. However, about one-third of the adenocarcinomas in the head of the pancreas had a periampullary location, while most neoplasms in other pancreatic lobes arose along the main pancreatic ducts. There was evidence of "leaking" of pancreatic juice through altered epithelium of main ducts, and this may have caused a marked periductal chronic inflammatory reaction.


Decreased retention of actinomycin D as the basis for cross-resistance in anthracycline-resistant sublines of P388 leukemia.

Sublines of P388 leukemia resistant to Adriamycin and daunorubicin were cross-resistant to actinomycin D in vivo and in vitro. The Adriamycin-resistant cell line was 1000-fold resistant to actinomycin D on 1-hr exposure in vitro and 370-fold resistant when exposed to the drug for 16 hr. The immediate binding of radioactive actinomycin D to sensitive and resistant cells was similar, and the uptake of the drug by the resistant cells was only about 27% less than the rate of uptake by sensitive cells. There was a dramatic difference in efflux of drug from sensitive and resistant sublines. Equivalent cytotoxicity of actinomycin D for the sensitive and resistant sublines was obtained at concentrations of the drug that resulted in approximately equivalent levels of net retention of actinomycin D (retained drug minus background levels of immediate binding of the drug to the cells). Incubation of cells in the presence of actinomycin D plus either Tween 80 or acridine orange incresed the rate of uptake and the percentage of actinomycin D retained by the resistant cells on short-term assays but did not reverse the resistance. It is concluded that these tumors must retain appreciable concentrations of actinomycin D for several hr in order to be killed. The anthracycline-resistant sublines are cross-resistant to actinomycin D by virtue of their inability to retain the drug.


Disordered nocturnal prolactin regulation in women with breast cancer.

Mean 24-hr prolactin concentrations were determined in 25 female control subjects, 16 women with benign breast masses, and 23 subjects with breast cancer. This evaluation performed before breast surgery revealed significantly decreased (p less than 0.02) nocturnal (12 a.m. to 7 a.m.) prolactin concentrations in 12 postmenopausal breast cancer subjects that contrasted with significantly increased (p less than 0.05) nocturnal prolactin levels in five luteal-phase premenopausal women with breast cancers. Prolactin concentrations in patients with benign breast disease were not significantly different from control subjects. Two of the premenopausal breast cancer patients had marked preoperative elevations in their mean 24-hr prolactin levels, and they were two of the three subjects who have since expired. Nocturnal prolactin secretion was significantly decreased (p less than 0.03) in four premenopausal breast cancer patients when they were studied 1 year after surgery; however, it remained the same in the eight postmenopausal breast cancer patients similarly evaluated. Although disordered prolactin regulation has been found in these women with breast cancer, its role in the etiology and progression of human cancer is still uncertain.


Twenty-four-hour preoperative endocrine profiles in women with benign and malignant breast disease.

Mean 24-hr growth hormone, luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone concentrations determined preoperatively in 16 women with benign breast masses and 17 patients with breast cancer were similar to those levels found in 25 age- and weight-matched control subjects. Mean 24-hr testosterone levels, however, were significantly elevated in women with breast cancer evaluated in the luteal phase of their cycles and were normal in postmenopausal breast cancer women. In addition, serum thyroid-stimulating hormone, thyroxine, cholesterol, and triglyceride levels were normal in these subjects. Plasma cortisols and urinary 17-hydroxysteroid excretion tended to be higher in both the benign and malignant breast disease group and probably reflected preoperative anxiety. Hence, we have found normal concentrations of a variety of endocrine and other biochemical agents that can stimulate breast tissue growth and/or have been previously reported to be disordered in women with breast cancer.


Estrogen receptor as an independent prognostic factor for early recurrence in breast cancer.

The usefulness of estrogen receptor measurements in primary breast tumors in the prediction of early recurrence was examined in a series of 145 patients. The absence of estrogen receptor in such tumors was associated with early recurrence independent of other known prognostic factors such as axillary lymph node status and tumor size.


Chemotherapy for adult acute nonlymphocytic leukemia with daunorubicin and cytosine arabinoside.

Fourteen of 21 adult patients (67%) with acute nonlymphocytic leukemia achieved a complete remission (CR) after receiving combination chemotherapy with daunorubicin and cytosine arabinoside (ara-C). Eight of the 14 CRs developed after a single course and four of 14 after two courses of induction therapy making initial hospitalization relatively brief (median, 38 days). Four of five patients greater than 60 years old achieved CR. The induction therapy was repeated monthly up to the dosage limits imposed by daunorubicin cardiotoxicity in an attempt to lengthen subsequent remission duration. The media duration of CR was 12 months which compares favorably with previously reported series. In this series, treatment with 3 days of daunorubicin and 7 days of ara-C proved to be a highly effective induction regimen for patients with acute nonlymphocytic leukemia regardless of age. The improved duration of CR may be a manifestation of the extent of initial leukemic cell-kill in successful induction and consolidation therapy rather than an effect of maintenance therapy cycles.


Methyl-CCNU versus methyl-CCNU and 5-fluorouracil in carcinoma of the large bowel.

5-Flourouracil (5-FU) and methyl-CCNU have demonstrated separate sensitivities in carcinoma of the large bowel. This study was an attempt to see if methyl-CCNU versus methyl-CCNU plus 5-FU would demonstrate different responses in advanced colorectal carcinoma. Forty-nine patients have been evaluated, 14 receiving methyl-CCNU and 35 receiving 5-FU plus methyl-CCNU. One partial response has been seen with methyl-CCNU alone in a patient with liver metastasis. Thirteen partial responses have been noted in patients treated with the two-drug combination. There was a significant difference in the median survival of the responders versus the nonresponders for the two-drug group. Side effects were expected: nausea and vomiting, leukopenia, and thrombocytopenia. Plasma carcinoembryonic antigen and urine arylsulfatase were measured in all patients and correlated well with response.


Characterization and responsiveness of the Madison 109 lung carcinoma to various antitumor agents.

The Madison 109 (M109) tumor was discovered in 1964 in the lung of a BALB/c mouse. This experimental carcinoma is maintained in vivo by sc passage in the right axillary region. When implanted im (5 X 10(5) cells) into the right hind leg of BALB/c mice for testing, the primary progresses with metastases to the lung, spleen, and liver. The metastases to the lung are visible within 3 weeks and result in the death of the host in about 35 days after tumor implant. Implantation of a lung nodule is tumorigenic and lethal. Pyran polymer therapy delayed the appearance of lung metastases, inhibited the growth of the primary tumor, and significantly increased the lifespan of BALB/c mice inoculated with the M109 tumor. No spontaneous regression has been observed and very few "no takes" have occurred in untreated BALB/c mice inoculated with at least 500 M109 cells. Of the 82 agents tested so far, the M109 model has selected active agents such as actinomycin D, adriamycin, daunorubicin, DNA, procarbazine, and pyran polymer. It has not shown sensitivity as tested to several standard therapeutic agents including cytosine arabinoside, BCNU, hydroxyurea, mechlorethamine, melphalan, triethylenemelamine, and vincristine.


Phase II trial of Baker's antifol in metastatic sarcoma.

Iv Baker's antifol (BAF) (250 mg/m2/day X 3 consecutive days) was administered to 34 patients with metastatic sarcoma. All patients had received extensive prior therapy including prior chemotherapy and had progressive disease at the start of the study. Liver and renal functions were normal in all patients. Of 29 patients evaluable for response, 25 demonstrated progressive disease and four had stable disease for periods of from 1 to 6 months. No objective responses were observed. The other five patients died from 3 to 12 days after initiation of therapy. Toxicity included myelosuppression of significant degree in nine patients, gastrointestinal effects of nausea and vomiting in seven, stomatitis in three, and dermatitis in four. Most toxicity was mild to moderate, although one drug-related death due to marked myelosuppression was seen. In conclusion, BAF is considered to be insignificantly active in the secondary treatment of metastatic sarcomas at the dose and schedule studied.


Passive immunotherapy for mouse leukemias with antisera of "directed" specificity: synergism with the action of cyclophosphamide.

Antileukemia sera with "directed" specificity are produced by immunization of rabbits with mouse leukemia cells admixed with normal antigen blocking (NAB) serum. Addition of NAB serum to the leukemia cells inhibits production of antibodies to normal cell components and directs specificity toward leukemia cell antigens. The resulting antileukemia serum (ALK-NABS) was not sufficiently potent to produce more than moderate therapy in the standard L1210 leukemia therapy assay. When given together with noncurative doses of cyclophosphamide (CTX), ALK-NABS acts synergistically. It is most effective when given early after injection of the leukemia cells and prior to injection of CTX. Daily repeated injections of a given dose are more effective than a single injection of that dose. Most important, small doses of ALK-NABS produce a significant prolongation of lifespan in conjunction with CTX. Results of therapy for BW-A leukemia with ALK-NABS in conjunction with CTX were negative.


Cellular response to treatment with 4-(3-(2-chloroethyl)-3-nitrosoureido)-cis-cyclohexanecarboxylic acid, a water-soluble nitrosourea derivative.

The lethal effects of 4-(3-(2-chloroethyl)-3-nitrosoureido)-cis-cyclohexanecarboxylic acid (cis-acid), a water-soluble nitrosourea derivative, were investigated on a human lymphoma cell line. The survival of asynchronous cells exposed to increasing concentrations of the drug was characterized by a threshold exponential curve (Do = 20 microgram/ml; Dq = 20 microgram/ml, 1 hour) similar to that of other nitrosourea derivatives. cis-Acid exerted its main killing effect on cells in early S and in late G2 phase. Cells in mid S and early G1 phase were tenfold more resistant. Changes in survival response as a function of cell cycle stage were reflected primarily by changes in the extent of the shoulder region of the survival curve. In contrast to other nitrosoureas, the lethal effectiveness of cis-acid in solution was stable and the drug could sterilize large numbers of cells in short periods of time. Another important major difference observed for cis-acid with respect to classic nitrosourea derivatives was the capacity of treated cells to recover from sublethal and potentially lethal damage. Our studies have shown that cis-acid is as effective in killing cultured human lymphoma cells as other nitrosoureas, but possibly with a mechanism different from that of these compounds. The major shortcoming noted for cis-acid, namely the capacity of treated cells to recover from drug-induced damage, is offset by the relatively long stability of its killing effect. This, and the fact that cis-acid can be administered in an aqueous solution, make this agent an appealing compound for clinical trials.


Preparation and antitumor evaluation of water-soluble derivatives of dichloro(1,2-diaminocyclohexane)platinum(II).

The structure of the antitumor agent, dichloro(1,2-diaminocyclohexane)platinum(II) (NSC-194814), was modified by replacing the chlorides with organic or inorganic anions. Eighteen new platinum complexes were so isolated and their antitumor properties against the L1210 leukemia in C57BL/6 X DBA/2 mice were evaluated. Most of the complexes were readily soluble in water and some had enhanced antitumor activity compared to the parent dichloro complex. In addition, increased solubility with retention of significant antitumor activity was obtained by oxidizing the parent dichloroplatinum(II) complex with halogen or peroxide to give two platinum(IV) complexes. Some previously reported platinum complexes with phosphorus, selenium, or tellurium electron donor ligands were also synthesized and assessed for antitumor action, but these did not show appreciable activity.


Chemotherapy for mustard-resistant ovarian adenocarcinoma: a randomized trial for CCNU and methyl-CCNU.

In view of the need for drugs which have antitumor activity in alkylating agent-resistant ovarian cancer or which might be more potent than standard agents, a randomized trial of two nitrosoureas, CCNU and methyl-CCNU, has been conducted. Of 31 evaluable patients who received at least one dose of CCNU (100 mg/m2 orally every 6 weeks), there were only two patients who experienced even transient improvement; 18 of these patients had two or more doses over a trial period of greater than or equal to 12 weeks. Of 26 evaluable patients who received at least one dose of methyl-CCNU (150 mg/m2 orally every 6 weeks), there was one patient who experienced transient improvement; this group included 14 patients receiving at least two doses and whose trial lasted greater than or equal 12 weeks. All patients had been previously treated with mustard-type alkylating agents. None of the responses were clinically useful. Thus, women with ovarian adenocarcinoma who have a history of prior chemotherapy are not likely to benefit from treatment with these nitrosoureas.


Whole body computed tomography in the diagnosis of abdominal and thoracic malignancy: review of 600 cases.

Whole-body computed tomography (CT) was performed on 600 patients with known or suspected malignancy at the National Institutes of Health. This new diagnostic modality utilizes a finely columnated X-ray source mounted opposite a computer-linked detector bank to generate anatomic cross-sectional images of the body part being scanned. CT proved valuable in defining many pathologic processes and was, at times, deemed the only test short of exploratory surgery that yielded diagnostic information. This latter benefit was most acutely emphasized in lesions affecting the retroperitoneum, adrenal glands, pelvis, liver, and immediate subpleural pulmonary parenchyma.


An in vivo stathmokinetic study of cell proliferation in human gastric carcinoma and gastric mucosa.

Cell production rates were studied in a group of eleven patients with carcinoma of the stomach using an in vivo technique with vincristine. In normal pyloric mucosa estimates ranged from 8 to 15 cells/1000 cells/hr, and in the carcinomas from 3 to 24 cells/1000 cells/hr. Because of the very large variation in the data, comparison between individual tumours and normal mucosa is not regarded as being worth-while at this stage. The implications of these results for previous human in vivo stathmokinetic experiments are also discussed.


Effects of uterine distension and oestradiol on cell kinetics in the endometrial epithelium of ovariectomized rats.

Cell kinetics in the uterine epithelium of ovariectomized rats were studied after uterine distension and/or an oestradiol injection, by cumulative 3H-TdR labelling and percentage of labelled mitoses (PLM). With both methods it was found that distension shortens the total cell cycle at the expense of G1 more than does oestradiol. Both treatments act in a cumulative manner since the greatest reduction in T c is observed after distension plus oestradiol. PLM curves showed that distension and/or oestradiol induce a 30% reduction S phase duration. The evolution of percentages of labelled cells and colchicine-blocked mitoses after these treatments confirms their additive effects and indicates that the mitogenic action of oestradiol is delayed compared to that of distension. It is suggested that these factors stimulate epithelial cell division in the uterus through partly different metabolic channels.


Cell loss from three established lines of the C3H mouse mammary tumor: a comparison of the 125I-UdR and the 3H-TdR-autoradiographic methods.

The 125I-UdR method for measuring cell loss from solid tumors has been reevaluated. The rate of tumor cell loss from three established lines (S 102F, S102S and Slow) of the C3H mouse mammary tumor was determined by the 125I-UdR method and the results were compared to the estimates for cell loss as determined by the combined approach of cellular 3H-TdR autoradiography and volumetric growth-rate determinations. This detailed comparison shows that the two methods complement each other but cannot substitute for one another because they give different quantitative information. The combined approach measures the flow of viable cells, as determined morphologically, from the proliferating compartment to the quiescent comparment, the quiescent compartment out of the tumor, etc., but does not evaluate the flow of degenerate cells or acellular (necrotic) debris. In contrast, the 125I-UdR method indicates the net flow of intact cells and/or dead cells as well as debris from the tumor as the 125I-labeled material is lost from the tumor, but gives limited internal information. thus, depending on the specific experiment, an investigator could choose one or the other of the methods to answer the question. Perhaps both would be desirable at times; however, in most cases, one could not substitute one method for the other. The data from the Slow tumors also indicate that in certain tumors, the quantitative information from the 125I-UdR method may be quite limited, i.e. the confidence limits within an experiment as well as the replication error between experiments may be high.


Comparison of growth characteristics of experimental tumours and derived cell cultures.

Cells from seven different rat tumours and a mouse sarcoma have been transplanted in syngeneic animals and were cultured in vitro. Tumours produced by inoculation of cultured cells in animals have been compared with the primary tumours. For the transplanted tumours, volume doubling times, T d, have been compared with doubling times, T d(cult), of cell numbers in cultures. Volume doubling times of the transplanted tumours generally decrease with increasing volume. At volumes of about 0-5 cm3, T d values range from 2-2 days to 10 days, while T d(cult) values ranged from 11 to 24 hr. A systematic correlation between T d and T d(cult) could not be established. During sequential transplantation of the tumours for many generations, as well as during continuous propagation of derived cell cultures, significant changes occurred which resulted in a decrease in the expression of differentiation characteristics in tumours.


An autoradiographic study of the 3H-uridine and 3H-thymidine incorporation in the regenerating mouse liver.

An autoradiographic study was made of the 3H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of 3H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of 3H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.


Grain count threshold dependence of FLM curve pattern and cell cycle parameters of hepatocytes in vivo.

FLM curves from hepatocytes of regenerating rat liver in vivo were compared at different grain count thresholds. Estimates of cell cycle phases derived from curves with thresholds decreasing from 15 to 1 grain (background 0-2 grains per nuclear area) revealed a prolongation of ts from 6-6 to 9-5 hr, at the expense of tG2M, and tG1, whereas tc remained constant. A similar pattern was observed in FLM curves at various threshold levels for hepatocytes localized in subunits of the liver lobule along the vascular axis from afferent to efferent pole. The shapes of these FLM curves indicated an intralobular gradient of reutilizable labelled material. The use of two different threshold levels is crucial for proper selection of FLM curves to evaluate cell cycle phases in regenerating rat liver: first, threshold to exclude the autoradiographic background, and a second one to avoid errors due to reutilization of labelled DNA precursors. Each threshold has its own implications for the estimation of cell cycle phases.


Replicon number and growth rate in fathead minnow cells cultured at 14 and 34 degrees C.

DNA fiber autoradiography was performed on cells of the fathead minnow cultured at 14 and 34 degrees C. Replicon growth rates were found to be about twice as fast at the higher temperature, but there was no appreciable difference in the number of replicons.


Estimation of the migration of thoracic duct lymphocytes to non-lymphoid tissues. A comparison of the distribution of radioactivity at intervals following i.v. transfusion of cells labelled with 3H, 14C, 75Se, 99mTc, 125I and 51Cr in the rat.

The distribution of radioisotopes in tissues was measured following i.v. injection of labelled thoracic duct lymphocytes into syngeneic rats. The rate of elution of an isotope from the labelled cells and the subsequent fate of the eluted isotope were shown to be the most important factors limiting the usefulness of such isotopes for measuring cell localization particularly in non-lymphoid tissues. Comparison of labelling procedures using [3H] and [14C]uridine, [3H] and [14C]leucine, [75Se]-L-selenomethionine, [99mTc]sodium pertechnetate and [51Cr]sodium chromate in vitro and [3H]thymidine in vivo showed that 51Cr had the fewest disadvantages in the present context. Using 51Cr-labelled cells, the radioactivity was measured in a wide range of non-lymphoid tissues, and estimates of cell traffic were obtained. In skin, for example, the results indicate a cell flux in the range of 10(4)-10(5) lymphocytes/gm/hr. Evidence is presented which suggests that the early substantial localization of labelled cells in the lung is not an artefact due to sequestration or embolization of traumatized cells but probably reflects a slow intravascular transit time through this capillary bed. The primary lymphoid organs, thymus and bone marrow were shown to include a subpopulation of lymphocytes which belong to the recirculating pool. The thymus always contained a greater concentration of radioactivity at 24 hr than all non-lymphoid tissues except liver and kidney (approx. 0-1% of the recirculating lymphocyte pool) and the bone marrow was capable of temporarily accepting a substantial proportion (approx.25%) of the injected cells.


Circadian rhythms of presumptive stem cells in three different epithelia of the mouse.

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with 3H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.


Serial transplantation, histology and cellular kinetics of a rat adenocarcinoma.

Data are reported on the growth, histology and cell population kinetics of a spontaneous mammary tumour in the rat at the 1st and 35-40th transplantation generation. With serial transplantation subcutaneously in isologous animals, the macroscopic growth characteristics of the tumour did not alter. The tumour had a volume doubling time of approximately 1.6 days at 400 mm3, increasing to about 7 days at 20,000 mm3. The histology, however, changed dramatically from that of a well differentiated papillary adenocarcinoma with a well defined vascular pattern and minimal necrosis to become by the 35th generation a poorly differentiated pleomorphic cellular tumour in which stromal elements and associated vascular supply were minimal and necrosis prominent. These changes were not manifested in the median cycle time of its cells, 16-18 hr, but by an increase in the growth fraction from 0.36 to 0.59 with a commensurate increase in the cell loss factor from 0.04 and 0.30.


Prediction of the complete growth pattern of human multiple myeloma from restricted initial measurements.

A method is presented for predicting the complete growth pattern of human IgG multiple myeloma from a few initial measurements of tumour cell number. This permits identification of patterns of undisturbed neoplastic growth in individual patients whose therapy cannot ethically be withheld for long periods. Since the unperturbed growth pattern is a necessary part of the information required for the prediction of individual patient response to alternative treatment schedules, the prospects for improved treatment of human multiple myeloma by optimal scheduling of drug administration are greatly increased.


Morphological alterations and functional changes of interhepatocellular junctions induced by bile duct ligation.

The effect of bile duct ligation on the intercellular junctions of hepatocytes was investigated. The features and the arrangement of the bile canaliculi and the zonulae occludentes alter concomitant to the increase of the intracanalicular pressure. The lumen of the bile canaliculi enlarges and the microvilli disappear. The array of the zonulae occludentes becomes irregularly shaped, the number of strands diminishes and interruptions of the strands occur. With peroxidase a leakage in the bile-blood barrier is detected. Furthermore a disappearance of gap junctions between the hepatocytes after bile duct ligation is observed. The present investigation shows that the zonulae occludentes are mobile structures which are changed by increased unilateral pressure. Due to their ultrastructural alterations, a leakage of the permeability barrier between physiological compartments is found.


Specific synaptic systems in reaggregated spherules from dissociated chick cerebellum cultivated in vitro.

Studies were performed on spherules of approximately 100-300 micrometer in diameter obtained from in vitro cultures of reaggregates of embryonic fragments of cerebellum from 10-12 day-incubated chick embryos, dissociated with trypsin and cultivated in a rotating shaker for a maximum of 21 days. The differentiated neurons within these spherules included a few Purkinje cells, many granule cells and type II Golgi cells, as well as many glial cells. Zones rich in synaptic knobs and other simple synaptic structures as well as complex synaptic systems with numerous active points of contact, were visible in various parts of the spherule. Typical "glomeruli" consisting of a varicosity of "rosette" joined to the dendritic claws of the granule cells, and "en marron" systems with perikarya of type II Golgi cells were easily recognised. The complete absence of extracerebellar afferents confirms that both the granule and Golgi cells are capable of making synaptic connections with afferents different from those normally formed by extracerebellar mossy or climbing fibres. The experimental findings confirm that the recipient neurons determine the specific synaptic pattern regardless of the nature of the afferents, and furthermore demonstrate that the clinging activity of the recipient neuron determines the synaptogenic behaviour of nervous pathways.


Electron microscopic study of the mandibular glands of Kalotermes flavicollis fabr. (isoptera; calotermitidae).

The mandibular glands of Kalotermes were examined in different castes. They show sexual dimorphism in the soldiers and primary reproductives, Moreover, in female soldiers and queens, mandibular gland cells contained numerous crystalline structures of mitochondrial origin. The role of these glands (secretion of saliva or pheromone) is discussed.


Fine structure of the intestinal epithelium of the colonial ascidian Botryllus schlosseri.

In the intestine of the filtering zooids of B. schlosseri three segments can be distinguished. In the intermediate segment the epithelium, which is encrusted by the ampullae of the pyloric gland, shows marked aspects of alteration. In the proximal and distal segments, ciliated mucous, vacuolated and endocrine-like cells are recognizable. Ciliated mucous cells, widely distributed along the intestine, possess the apical region filled with numerous mucous granules, which are extruded with merocrine modality. Variations in morphology of the granules are visible especially between cells of different regions. Vacuolated cells appear involved in absorptive function. They are characterized by developed microvilli, numerous apical small vesicles and great supranuclear vacuoles containing heterogeneous material. The vacuolated cells of the proximal segment resemble the gastric vacuolated cells of B. schlosseri. The vacuolated cells of the distal segment show many morphological similarities with protein absorbing cells of various animals for the presence of a giant vacuole and an apical network of vesicles and tubules with fuzzy coating on the luminal face. The intestinal endocrine-like cells are rare and characterized by strongly electron dense granules distributed in all the cytoplasm, but predominantly in the basal region.


The time sequence of response of the prolactin cells of the freshwater teleost, Poecilia reticulata, to an altered environmental salinity.

The time sequence of the ultrastructural changes in the prolactin cells of the adult guppy, Poecilia reticulata, was studied in freshwater fish transferred to 1/3 seawater for 1, 4, 11 and 28 days. The morphological changes were slight and only detectable by quantitative (morphometric) procedures. The secretory granules showed a "biphasic' response to the altered environmental salinity, but the volume density of the Golgi apparatus progressively declined throughout the experiment. After an initial decrease in the volume density of the RER, that organelle regained its original prominence by 11 days in 1/3 seawater. The volume density of the nucleus was markedly higher in fish 28 days after transfer to 1/3 seawater than in any other group. Cell volume estimations showed that a shrinking of the cytoplasm rather than a swelling of the nucleus accounted for the high volume density figure. The changes in the volume density of the mitochondria closely paralleled those of the RER. Profiles of exocytosed granules were rarely found in any of the groups, but were least frequent in fish kept in 1/3 seawater for 28 days, when dense (lysosomal?) bodies were most abundant. The quantitative methods high-lighted some discrepancies in the rate and magnitude of the changes shown by some organelles and have led the authors to suggest that during adaptation of fish to dilute seawater, synthesis and release may not be coupled processes in the prolactin cell of P. reticulata.


Microtubules in myelinated and unmyelinated axons of rat sciatic nerve.

Tannic acid in glutaraldehyde was used to stain microtubules in myelinated and unmyelinated axons of rat sciatic nerve. In the majority of areas the tannic acid failed to penetrate the unmyelinated axons whilst penetrating neighbouring myelinated axons, suggesting a difference in the ability of the two types of nerves to exclude tannic acid. Where tannic acid had penetrated the unmyelinated axons the 13 protofilament substructure and size of the microtubules appeared identical to those seen in the myelinated axons.


Fine structure of capillaries in the conus papillaris of the limbless lizard, Ophisaurus apodus (anguidae, lacertilia).

The conus papillaris of Ophisaurus apodus consists of blood vessels and pigment cells. The capillary walls are formed by endothelial cells, scarce pericytes and basal laminae. The cell bodies are attenuated and the plasmalemma of their luminal and abluminal surfaces forms microvilli. The perivascular space is well developed, containing nerve fibers and their terminals. Similar localization and ultrastructure of avian pecten oculi and lacertilian conus papillaris suggest homology of these structures.


Intrapulmonary neuro-epithelial bodies in newborn rabbits. Influence of hypoxia, hyperoxia, hypercapnia, nicotine, reserpine, L-DOPA and 5-HTP.

Neonatal rabbit neuro-epithelial bodies (NEB) were investigated under various experimental conditions with light microscopy, microspectrography, morphometry and electron microscopy. (1) Hypoxia causes a decreased amine fluorescence intensity and an increased secretory exocytosis of dense core vesicles (DCV). Otherwise the NEB appear structurally normal. (2) Hypercapnia also produces a decreased fluorescence and an increased exocytosis; ultrastructurally, however, the dense core of DCV fragmentizes. (3) Hyperoxia does not appear to affect significantly either fluorescence or exocytosis. (4) The uptake of biogenic amines such as 5-HTP and L-DOPA was demonstrated by fluorometry and electron microscopy. (5) Reserpine, on the other hand, provokes an amine depletion with a decrease of the NEB fluorescence and an ultrastructural palor of the DCV. (6) Intratracheally administered nicotine is accompanied by a decreased fluorescence and a distinct exocytosis of fragmented DCV. The reaction of NEB to hypoxia and hypercapnia suggests that these corpuscles could be intrapulmonary chemoreceptors (in addition to the classically known central and peripheral chemoreceptors), inducing a reflex reaction through the liberation of DCV at the corpuscular sensible nerve endings and via the CNS. In addition, they may subserve a local intrapulmonary effect by modulating directly the hypoxic and hypercapnic pulmonary vasoconstriction and thus the V/Q ratio.


Stereoscopic scanning electron microscopy of the red pulp of dog spleen with special reference to the terminal structure of the cordal capillaries.

In order to obtain direct morphological information about the three-dimensional fine structure of the splenic terminal vascular bed, especially the terminating mode of the cordal capillaries, stereoscopic scanning electron microscopy of perfusion-fixed and freeze-fractured red pulp of a normal dog spleen was undertaken. An improved method of perfusion-fixation was utilized in which the hydrostatic pressures of the splenic artery and vein were maintained at approximately the same levels as those in the living state. Stereoscopic observations of scanning electron micrographs clearly demonstrated the three-dimensional fine architecture of the splenic sinuses, the spongy cordal reticular tissue and the intracordal vasculature. The cordal capillaries terminate in the labyrinthine cordal space according to a certain mode in which the walls of the terminals are transformed into a meshwork structure continuous with the cordal reticular tissue owing to an increase in number and size of fenestrations. No evidence could be detected to prove or suggest any direct continuity of the capillary end with the splenic sinus. These results strongly support the concept of an "open circulation", at least in the red pulp of the dog spleen, with the possibility of a "functionally closed circulation" under some physiological conditions.


Electron microscopic study of terminal degeneration in the anterodorsal thalamic nucleus of the cat.

The ultrastructure and synaptic organization of the anterodorsal nucleus (AD) of the thalamus were investigated under normal and experimental conditions. The large glomeruli are composed of an extensive central dendrite, probably arising from a projecting neuron, and of various terminal boutons. Besides the typical small bouton (RS) filled with round vesicles, two specializations of the large bouton (RL) containing round vesicles are found. The larger one (RL1) is characterized by a looser arrangement of synaptic vesicles and many mitochondria; it undergoes dark degeneration following unilateral lesions of the mamillary body in both AD. The intermediate type (RL2) is distinguished by a denser arrangement of vesicles; it undergoes dark degeneration following lesions of the midline nuclei of the thalamus. The two types of terminal boutons (F1 and F2) which contain flattened or pleomorphic vesicles do not degenerate following lesions of the mamillary body and midline nuclei; they belong to Golgi type II interneurons.


An ultrastructural analysis of the gametes and early fertilization in two bivalve molluscs, Chama macerophylla and Spisula solidissima with special reference to gamete binding.

An ultrastructural investigation of the gametes and their interaction during the early events of fertilization in molluscs has been performed. A gamete binding event involving large numbers of sperm has been identified and examined in detail. The surface of the oocyte is projected into numerous microvilli which extend through the vitelline envelope. Tufts of fibrillar material radiate from the tips of these microvilli, forming a layer external to the vitelline envelope. The acrosomal vesicle of the mature spermatozoon contains two major components, which function differently during fertilization. The vesicle is indented at its adnuclear surface, constituting a preformed acrosomal tubule. This tubule does not elongate during the acrosome reaction. Completion of the reaction results in the formation of an extracellular coat, derived from on component of the acrosomal vesicle, on the anterior surface of the sperm. Sperm-egg binding is accomplished by an association of the extracellular coat on the reacted sperm and the fibrous tufts on the tips of the microvilli of the oocyte. Evidence that gamete membrane fusion occurs by fusion of the acrosomal tubule and a microcillus is presented. These observations provide a generalized pattern of molluscan fertilization.


The role of the haemocytes of Clitumnus extradentatus in haemolymph coagulation.

Light and electron-microscopic observations of the blood cells (haemocytes) of the stick insect Clitummus extradentatus in vitro showed that two morphologically distinct cell types, the cystocytes and granular cells are involved in haemolymph coagulation. Both these cell types contain a variable number of electron-dense granules which upon release cause progressive coagulation and precipitation of the haemolymph. In the cystocytes this release is extremely rapid (30-60s) while in the granular cells it only occurs after 1-2 min in vitro. The role of these cells in haemostasis is discussed together with the possible significance of the involvement of two cell types in the coagulation process.


Ultrastructural tracer studies on the permeability of the Malpighian tubule of the pill millipede, Glomeris marginate (Villers).

The electron-dense tracers ferritin, and iron-dextran, and the protein horseradish peroxidase, have been used to investigate the ultrastructural basis of permeability in the upper and lower segments of the Malpighian tubules of Glomeris marginata. All these materials were able to cross the basal lamina and enter the tubule lumen of the upper segment, and it was established that horseradish peroxidase was able to enter the channels which interrupt the apical junctions. In the upper segment, ferritin, iron-dextran, and horseradish peroxidase are taken up and accumulated within intracellular vesicles. In the lower segment ferritin and iron-dextran enter the cells but become generally distributed over the cytoplasm, as well as entering membrane-bounded vacoules. The behaviour of horseradish peroxidase could not be assessed by owing to the presence of endogenous peroxidase activity in the cells. After fixation by direct application of glutaraldehyde to the undissected tubules, the extracellular spaces contained large numbers of membrane-bounded vesicles. The significance of these observations is discussed in relation to the physiological activities of the tubules.


The ultrastructure of the sinus gland of Gammarus oceanicus (crustacea: amphipoda).

The sinus gland of Gammarus oceanicus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.


A morphological study of the circadian cycle of the pineal gland of the rat.

To seek a morphological expression of circadian rhythm, we investigated cytologically pineal glands taken from rats every 2 to 4 h under a lighting regime of 12 h of illumination (6:00 to 18:00) and 12h of darkness. The changes in the number of synaptic ribbons and ribbon fields was observed by electron microscopy. The number of these intracellular elements was greatest at 2:00 and lowest at 14:00, the difference being statistically significant. There were no significant local differences in numbers with respect to the part of the pineal gland examined. The data are similar to those of Vollrath from the pineal gland of a guinea pig, and seem to confirm a circadian function in the pineal gland in mammals.


Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo. II. Early ultrastructural modifications correlated to the biochemical lesion induced by fluoroacetate.

Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slighly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.


Scanning electron microscopic investigations of olfactory epithelium in the chick embryo.

This paper describes maturation of the surface of the olfactory epithelium of the superior nasal concha of chick embryos from the seventh day of incubation till hatching. From the earliest stages investigated onward, the apical surface of the olfactory epithelium is characterized by variability in the number and configuration of surface protrusions. The growth of olfactory cilia is described and the significance of regional differences in their density is discussed in relation to regional variations in the sensitivity of the epithelium to odorants. The maturation of the respiratory epithelium covering the inferior nasal concha is also described.


Structure of sinuses in the human lymph node.

A casting technique has been employed to display in three dimensions, the lymphatic microcirculation within the human lymph node. The casting compound filled the marginal sinus, and diffusely permeated the cortical lymphoid parenchyma. However, deep within the lymph node in the medullary region, the medium remained within the limits of the sinus walls. The casts showed well-defined channels appearing similar to vessels. These converged into larger vessels, which drained into efferent lymphatics leaving the node at the hilus. Electron microscopic examination showed that the outer wall of the marginal sinus and the trabecular side of trabecular sinuses had an intact, continuous endothelium with a basement membrane. However, gaps were present in the inner wall of the marginal sinus, as well as in the parenchymal wall of the trabecular sinus. In the medulla, the sinuses were lined by endothelial cells which appeared similar to macrophages. The sinus lining was incomplete and possessed numerous perforations. These observations indicated that sinus walls adjacent to connective tissue served as a barrier to cell movement, but those adjacent to a large lymphoid cell population had gaps, with cells in apparent transit between sinus lumen and parenchyma.


Studies on the ovotestis of the slug agriolimax reticulatus (Müller). 2. The epithelia.

The follicle cells, nurse cells and germinal epithelia, which are closely associated with the oocyte of Agriolimax reticulatus (Müller) during its development in the ovotestis, have been studied using light and electron microscopy. The various secretory, digestive and phagocytic activities of these cells have also been investigated using electron cytochemical tests for oxidisable polysaccharide, acid phosphatase and electron-opaque tracer molecules. The oocyte lies initially between the germinal epithelia and a layer of nurse cells but, as oocyte vitellogenesis proceeds, it becomes encapsulated by a layer of follicle cells. Both the follicle and the nurse cells are active in secretion and digestion and contain Golgi apparatus, granular endoplasmic reticulum and acid phosphatase-rich digestive vacuoles. The significance of these activities is discussed in relation to oocyte vitellogenesis, secondary envelope formation and the digestion and recycling of cellular material.


Studies on transmembrane and paracellular phenomena in the foot of the slug agriolimax reticulatus (Mü).

Using the enzyme peroxidase and ionic lanthanum as tracers, paracellular uptake has been demonstrated in the foot of the slug Agriolimax reticulatus (Mü). Both tracers appeared to pass between adjacent foot epithelial cells and were demonstrated in the zonula adhaerens, the septate desmosomes, and the intercellular spaces which occur beneath the septate junctions. Ferritin, a somewhat larger tracer, was excluded from all these sites. Ionic lanthanum was not normally pinocytosed in short incubation times. The epithelial cells could be induced to endocytose this marker, however, when combined with a variety of proteins. The implications these findings have on the uptake of molluscicides is discussed.


Ultrastructural localization of juvenile hormone biosynthesis by insect corpora allata.

The conversion of exogenous 3H-farnesenic acid to 3H-methyl farnesoate and 3H-C16 juvenile hormone (JH) has been followed in the corpus allatum (CA) cells of the desert locust Schistocerca gregaria by means of electron microscopic autoradiography. Aerobic and anaerobic 'chase' incubations have been used to modify the quantities of these three compounds within the CA cells. Under all incubation conditions, radiolabel is found associated almost exclusively with the subcellular membrane systems - smooth endoplasmic reticulum (SER) and Golgi elements - and with the mitochondria. CA cells are probably similar to vertebrate steriod-synthesizing cells in that the secretory product is synthesized in the SER and mitochondria. Radiolabel was found to be present in all cells of the CA suggesting that all cells are capable of at least the final two stages of JH biosynthesis (the esterification and epoxidation of 3H-farnesenic aid). This indicates that JH biosynthesis may be regulated through changes in the biosynthetic capabilities of individual cells rather than through changes in the total number of cells engaged in biosynthesis. Radiolabel was not observed to be associated with any distinctive cellular product, a result which provides additional evidence for the suggestion that the release of JH from the CA is governed by laws of simple physical diffusion.


Endocrine cells in the human fetal small intestine.

In this report we describe the time of appearance and ultrastructural features of enteroendocrine (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9--10 weeks. They were arbitrarily termed "primitive" and "precursor" cells. As in all fetal EECs, the pale cytoplasm of the "primitive" cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200-330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the "precursor" cells are larger, averaging up to 1 micron in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed "transitional" cells. These have two populations of SGs; one resembles the SGs of the "precursor" cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D1 cells were identified by 12 weeks of gestation. The "primitive", "precursor", and "transitional" cells may represent sequential developmental precursors of adult type EECs.


On the yolk sac of the cat. II. Erythropoietic phases, ultrastructure of aging primitive erythroblasts, and blood vessels.

The phase of primitive erythropoiesis in the feline yolk sac lasts from the 14th to the 20th day after malting. The globular nucleated primitive erythroblasts are formed extravascularly to some extent, but they can be clearly distinguished from the endoderm. They do not undergo a denucleation and are still present in the circulating blood on the 45th day. Aging primitive erythroblasts are characterized by a loss of polysomes, by the appearance of long intracytoplasmic electron-lucent channels, and by a nuclear pyknosis which can turn into a karyolysis. Definitive erythropoiesis begins around the 17th day but, even by the 19th day, it is not particularly prominent. It ends around the 45th day. It is almost exclusively intravascular. The distinction of immature primitive erythroblasts from erythroblasts of the definitive series is difficult, because it is based upon only slight differences in the heterochromatinization, in the nuclear-cytoplasmic ratio, and in the organelle content of the cells. In the definitive series, the nuclear divisions follows the law of the rhythmical halving of the nuclear volume. The cells exhibit more clearly identifiable maturation stages here, and the 'checkerboard nucleus' is more distinct. The vascular endothelium is largely attenuated and moderately fenestrated; it lacks a distinct basement membrane. Organelle-rich adventitial cells are found in close apposition.


Protein turnover in the smooth muscle cell of the aortic tunica media as visualized by radioautography.

The turnover of newly formed proteins in the aortic myocyte was studied by means of quantitative light and electron microscopic radioautography. Adult and young mice were sacrificed at long intervals between 4 h and 108 days after one injection of tritiated leucine. The results showed that secretory activity, important in the post-natal myocyte, persists noticeably in the mature myocyte. The similarity in sedentary protein turnover, noted in both the thoracic and abdominal aortic segments of the adult and young animals, seems to be expressed by the same labelling patterns of the myoplasma. Turnover of exportable protein, which varies with age and anatomical level, appears to be related to the higher rate of elastin synthesis in the thoracic aorta of the young animal.


DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver.

Ultrastructural analysis of M-band from nuclei of rat liver showed small amounts of chromatin, fragments of inner nuclear membrane, some amorphous nuclear material, and nucleopores. The outer nuclear membrane with its associated ribosomes was removed by Sarkosyl during the preparation of M-band sample. Morphological features of nucleopores and the inner nuclear membrane were confirmed by freez-fracture technique. The gross chemical composition of the M-band was similar to that of nuclear-membrane fractions prepared by other techniques. The M-band contained the greatest proportion of newly-labeled DNA and also supported DNA synthesis in vitro. Electron-microscopic autoradiography of the M-band showed localization of silver grains of thymidine-3H presumably over newly synthesized DNA. The DNA synthesis could not be attributed to spurious attachment of DNA polymerase to M-band during its isolation. It was partially removed from the M-band by treatment with 0.5 M KC1, phospholipase A or C; and completely, by the action of pancreatic DNase. DNA synthesis was greater in M-band fractions isolated from nuclei of 24-hour regenerating liver.


The fine structure of the interrenal cells of the duck (Anas platyrhynchos) with evidence for possible exocytotic release of steroids.

The duck interrenal cell possesses ultrastructural characteristics common to other steroid-secreting cells. Lipid droplets and mitochondria are abundant and lie principally at the apical end of the cell. Lipid droplets are not membrane-limited. Cisternae of smooth endoplasmic reticulum that are occasionally continuous with the less abundant rough endoplasmic reticulum are a prominent feature of the interrenal cell. Tubular profiles of rough endoplasmic reticulum often lie tangentially to mitochondria and ribosomes are either free, grouped in polyribosomal clusters, or bound to the endoplasmic reticulum. Mitochondria possess tubular cristae in the inner regions of the gland and frequently contain a paracrystalline array of small 10nm (o.d.) tubules and less frequently a hexagonal array of 40 nm trilaminar rings. Other cytoplasmic components include dense bodies, residual bodies, microtubules, microfilaments and specialized single membrane-bound vesicles. Gap junctions, intermediate junctions and interdigitating processes constitute the main intercellular associations. No tight junctions were identified. The single membrane-bound vesicles which are occasionally filled with a low electron-dense, lipid-like material form septate-like "junctions" with the plasma membrane. The septa bridge an intracellular gap of 15-17 nm. The vesicles are usually located near the subendothelial space at the basal and basilateral regions of the cell. Occasionally, vesicles fuse with the plasma membrane. It is suggested that these vesicles represent morphological evidence for the exocytotic release of steroid hormones.