Carbohydrate compositions of normal, spontaneously transformed, and virally transformed cells derived from BALB/c mice.

Carbohydrate compositions of chloroform: methanol-soluble and -insoluble complex polysaccharides have been studied in cell lines derived from BALB/c mice. The cells used in these studies include normal cells, spontaneous and viral transformants that cause tumors that regress, and spontaneous and viral transformants that cause progressively growing tumors that kill immunocompetent BALB/c mice. The carbohydrates were determined by gas-liquid chromatography. Some of the transformed cell lines compared with normal cells have altered carbohydrate compositions, including decreased sialic acid levels and decreased N-acetylgalactosamine in chloroform: methanol-soluble material. Significant decreases in total carbohydrates of chloroform: methanol-soluble material were also observed in some of the transformed cells. However, these changes alone do not predict cancer. Transformed cell lines that cause progressively growing tumors tend to have fewer alterations in carbohydrates of complex polysaccharides than lines causing tumors that regress.


Combination versus sequential five-drug chemotherapy in metastatic carcinoma of the breast.

Patients with metastatic or recurrent adenocarcinoma of the breast were randomized to weekly combination chemotherapy, intermittent combination chemotherapy, or single-drug chemotherapy administered sequentially. Patients receiving weekly combination therapy were more likely to respond than those receiving single-drug therapy (5-fluorouracil). The median survival for either group treated with combination therapy was double that of patients on sequential therapy. One-fourth of the patients had a prolonged survival (greater than 75 weeks), regardless of therapy.


Metabolism and metabolic effects of 8-azainosine and 8-azaadenosine.

8-Azainosine (8-aza-HR) is of interest because of its activity against experimental tumors. Metabolic studies in cell cultures were performed with 8-aza-HR and with the structurally related nucleoside, 8-azaadenosine (9-beta-D-ribofuranosyl-8-azaadenine) (8-aza-AR), which has a lower degree of antitumor activity than does 8-aza-HR. In H. Ep. 2 cells and in Ca755 cells, both 14C-labeled nucleosides were metabolized to nucleotides of 8-azaadenine (8-aza-A) and 8-azaguanine (8-aza-G) and incorporated into polynucleotides as 8-aza-A and 8-aza-G. 8-Aza HR was incorporated primarily as 8-aza-G, whereas 8-aza-AR was incorporated about equally as 8-aza-A and 8-aza-G. In H. Ep. 2 cells, the extent of incorporation of 8-aza-HR as 8-aza-G was about one-half that found when [14C]-8-aza-G was the precursor. In the H. Ep. 2/FA/FAR cell line, 8-aza-AR and 8-aza-HR were metabolized similarly, in that both were incorporated into polynucleotides principally as 8-aza-G; apparently, in this cell line which is deficient in adenosine kinase and adenine phosphoribosyltransferase, 8-aza-AR is metabolized by conversion to 8-aza-HR. A cell line (H. Ep 2/8-aza HR), which was resistant to 8-aza-HR but sensitive to 8-aza-AR and which retained hypoxanthine (guanine)-phosphoribosyltransferase activity, metabolized 8-aza-HR to only a small extent. However, in this cell-line, 8-aza-AR was more extensively metabolized and was incorporated primarily as 8-aza-A. The failure of these cells to convert 8-aza-AR or 8-aza-HR to 8-aza-G indicates that the basis for resistance may be a change in the substrate specificities of the enzymes of guanosine monophosphate synthesis such that these cells no longer effectively convert 8-azainosine monophosphate to 8-azaguanosine monophosphate. 8-Aza-AR was a potent inhibitor of purine synthesis de novo, but 8-aza-HR, at concentrations much higher than the inhibitory concentration of 8-aza-AR, did not inhibit this process. In H. Ep. 2 cells, 8-aza-HR blocked the conversion of orotic acid to uridine nucleotides and caused an accumulation of orotidine. This inhibition of pyrimidine biosynthesis apparently does not contribute significantly to the cytotoxicity of 8-aza-HR because uridine provided no degree of reversal of its inhibition of the growth of cell cultures.


Malignant transformation and viral replication of rat bone and muscle cells after in vitro infection with rat-adapted murine sarcoma virus (Moloney).

Isolated bone and muscle cells from rat fetuses were infected with the rat-adapted osteosarcomogenic murine sarcoma virus (Moloney) for examination of malignant transformation and viral replication. After the infection, the bone cells underwent an unusual transformation characterized by two patients in the focus formation. In the early transformation (Transformation I), foci consisting of only morphologically altered cells appeared, but they soon disappeared. Focus formation of this type continued for subsequent cell passages. In the late transformation (Transformation II), typical foci of malignant cells were formed with rapid multiplication. The cells in both types of transformation produced sarcomogenic as well as leukemogenic viruses. They contained C-type particles showing varying morphology and dimensions. Distinct bone cell tropism of osteosarcomogenic murine sarcoma virus (Moloney) was shown for three in vitro passages through rat bone cells. The infected muscles cells produced Transformation I foci at an incidence lower than did the bone cells, being mostly nontransformed. The virus yielded from these cells was predominantly leukemogenic, consisting of typical C-type particles. Evidence presented suggests that the tissue dependency with replicating ability of murine sarcoma virus and murine leukemia virus present in osteosarcomogenic murine sarcoma virus (Moloney) preparation is responsible for exhibiting the tissue tropism of this virus.


Selective lethal effect of supranormal temperatures on human neoplastic cells.

The effects of supranormal temperatures upon normal human cells (derived from normal adult and embryonal tissues) and neoplastic human cells (derived from biopsies of malignant tumors) have been quantitatively studied in tissue culture. Melanoma cells have been compared with melanocytes derived from fetal uveas. Colon carcinoma cells have been compared with embryonal intestinal epithelial cells. Malignant neuroepithelial cells derived from a teratocarcinoma of the ovary have been compared with neuroepithelial cells derived from fetal brain. Fibrosarcoma cells have been compared with normal adult fibroblasts. All cells defined as neoplastic have produced malignant tumors when injected into nude thymus-deficient mice at doses of 1 X 10(7) cells or less. Exposure to temperatures of 42.5-43.0 degrees for 4 to 8 hr has been shown to have significantly greater lethal effect on the tumor cells than on the nonneoplastic cells.


Molecular structure of benzo(a)pyrene 4,5-oxide.

An X-ray crystallographic study of benzo(a)pyrene 4,5-oxide, a metabolite of the carcinogen benzo(a)pyrene (BP), as given information on the geometry of this molecule. The carbon skeleton of BP itself has been shown by others to be early planar; the planarity of the carbon skeleton has been shown by this work to be perturbed very little by epoxidation of the 4,5-double bond. Epoxidation has, however, increased the double bond character of C-11--C-12, C-9--C-10, and C-7--C-8. The hydrogen atom on C-3 points directly toward the oxygen atom of another molecule. This C--H... O interaction, although weak, suggests that C-3 might be slightly acidic. An analysis of the experimentally determined bond lengths indicates that, after the highly reactive epoxide ring, the most reactive positions are at C-1, C-6, C-7, C-11, and C-12. The oxide ring of BP, unlike that for the K-region oxide of 7,12-dimethylbenz(a)anthracene, is symmetrical (with C--O distances equivalent within experimental error). The C--O distances are longer than those found in most oxides, including those in 7,12-dimethylbenz(a)anthracene-5,6-oxide. Thus it has been shown that the oxide rings of the K-region oxides of the two potent carcinogens BP and 7,12-dimethylbenz(a)anthracene are not similar in dimensions.


Histogenetic and morphological in vivo and in vitro data concerning androgen-estrogen-induced scent gland tumor in the Syrian hamster.

The scent gland tumor of the Syrian hamster is induced by exogenous androgen and estrogen. Microscopic nodules are induced normally in old males by endogenous androgen. The histogenesis of the scent gland tumor is complex and not completely understood. In this study microscopic preneoplastic nodules and macroscopic tumors were studied by light and electron microscopy, and the macroscopic tumors were grown in tissue culture on collagen-coated coverslips and on sponge foam matrices by the organ culture method. The cultures were fed with an unfiltered fetal calf serum-bovine serum ultrafiltrate medium, which contained endogenous androgen-estrogen, 110-100 pg and could maintain growth without additional androgen-estrogen. Exogenous androgen-estrogen was also added to some cultures. Scent gland tumors grown in organ culture contained cells of two shapes, spindle and ovoid arranged in cords. Cultures on coverslips showed radiating outgrowths of spindle cells suggesting either mesenchymal or Schwann cells. By electron microscopy, both in vivo and in vitro preneoplastic and tumor samples contained cells with segments of basal lamina, micropinocytotic vesicles, and junctional complexes. These features were similar to those of poorly differentiated experimental malignant rat schwannomas maintained in similar in vitro systems. Tumors grown in vivo and in vitro were associated with collagen fibrils with a periodicity ranging from 400 to 1075 A. The evidence reported in this paper suggests that one component of the scent gland tumor is an androgen-estrogen-induced poorly differentiated schwannoma.


Carcinogenic activity of a chlorinated polyether polyurethan.

On the basis of the results of an earlier study, a particular polyurethan sample (Y-238) was selected for further evaluation of its carcinogenic potential. This sample was subjected to physical and chemical tests for elucidation of its chemical structure, molecular weight, and molecular weight distribution. Additional biological tests were conducted on male NBR rats by implanting various quantities of the sample i.p., while others received an intrabronchus implant. Tumors, assessed histologically as malignant, were observed following both routes of implantation. The most common neoplasms of the pulmonary site was epidermoid carcinoma, while fibrosarcoma was the most common neoplasm in the peritoneal cavity. Data from the i.p. implantation suggested a dose-related incidence of cancers.


Establishment of a cell line (Co-115) from a human colon carcinoma transplanted into nude mice.

A human colon carcinoma cell line, Co-115, has been established in vitro from solid xenografts maintained in nude mice and subcultured for 95 passages. Co-115 cells grow in vitro as tightly packed, epithelial-like colonies, have a doubling time of about 36 hr, have a relatively low plating efficiency in agar, and release significant amounts of carcinoembryonic antigen to the culture medium. Their epithelial nature has been confirmed by ultrastructural examination. The injection of Co-115 cells into nude mice reinduced the formation of solid tumor masses that could be retransplanted and showed a morphology comparable of that of the original xenograft.


A factor responsible for increases in ornithine decarboxylase activity in the livers of tumor-bearing mice.

Ornithine decarboxylase activity in the livers, spleens, and kidneys of tumor-bearing mice changed markedly during tumor growth. These changes in enzyme activity were not due to infiltration or metastasis of tumor cells in these organs. After i.p. inoculation of Ehrlich tumor cells, enzyme activity in the liver and spleen increased remarkedly, reaching a peak in 4 to 6 days and then quickly decreasing. Conversely, activity in the kidney, which was very high in normal mice, decreased markedly during tumor growth, nearly reaching zero on Day 6 and remaining very low until death. Upon injection of a cell-free homogenate of Ehrlich tumor or cell-free ascites fluid, enzyme activity in the liver and spleen also increased markedly, but that in the kidney did not change. These increases in activity were not due to the effects of living tumor cells. Similar increases in enzyme activity were also observed in the livers of mice given injections of homogenates of Sarcoma 180 or Act. S tumor, or plasma from tumor-bearing mice, but not in the livers of mice given injections of homogenates of various nontumorous tissues, such as liver, kidney, spleen, muscle, regenerating liver, and fetus, or plasma obtained from normal mice. A similar increase in enzyme activity in the liver after injection of a cell-free preparation of tumor cells was observed in hypophysectomized and adrenalectomized mice; thus, these endocrine systems are probably not involved in the increase in enzyme activity in the livers of tumor-bearing mice.


Stimulated DNA synthesis in mouse epidermal cell cultures treated with 12-O-tetradecanoyl-phorbol-13-acetate.

Exposure of mouse epidermal cells in culture to 12-O-tetradecanoyl-phorobol-13-acetate (TPA) results in an initial inhibition of DNA synthesis for 24 hr followed by a 5- to 10-fold stimulation at 72 to 96 hr. A corresponding increase in mitotic rate also occurs at 72 to 96 hr. These responses occur when TPA is continuously present in the medium or if the exposure is as short as 1 hr, but the degree of stimulation was dependent on dose and duration of exposure. Sensitivity to TPA varied with the length of time the cells were in culture prior to treatment. TPA treatment also produced an alteration in morphology from clearly epithelial to a more fibroblastic type. These biochemical and morphological effects did not occur after treatment of epidermal cells with either phorbol-13,20-diacetate or phorbol. Primary dermal fibroblasts in culture did not respond to TPA in this manner, but a line of cultured liver epithelial cells was slightly stimulated by the promoter. This system appears to be a sensitive in vitro model for detecting the hyperplasia-inducing effects of phorbol esters and should be used for mechanistic studies and bioassay.


Potentiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea by amphotericin B in murine ependymoblastoma.

This paper reports the potentiation of the therapeutic effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by amphotericin B (AMB) in s.c. transplanted murine ependymoblastoma 01B111. The rate of 2-month cures was 6% when tumors were treated 10 to 12 days after transplantation by a single 6 i.m. injection of 2.5 mg of CCNU per kg and reached 15% with 10 mg of CCNU per kg. When 25 mg of AMB per kg were given i.p. 10 hr prior to CCNU, the respective figures increased to 18 and 58%, the differences being significant at 5 and 1%. A single dose of 25 mg of AMB per kg given alone did not affect tumor growth. Radioactivity per g of tumor was measured 30, 60, and 120 min after injection of [14C]CCNU in total tissue, chloroform:methanol (2:1, v/v) extract, and CCNU isolated by thin-layer chromatography. No difference was found between animals treated with 25 mg of AMB per kg and controls. The inhibition of DNA synthesis, measured 24 hr after the administration of CCNU and 2 hr after the injection of [3H]thymidine, was almost optimal with 5 mg of CCNU per kg. The inhibition caused by 1 mg of CCNU per kg was not enhanced by AMB. Thus, the potentiation of CCNU by AMB does not seem attributable to an increased permeability of the tumor to CCNU or to an enhancement of the inhibition of DNA synthesis, at least in murine ependymoblastoma.


Comparison of antitumor activities of pancreatic ribonuclease and its cross-linked dimer.

The cross-linked dimer of bovine pancreatic RNase (M.W. 28,000) is significantly more effective than the monomer in inhibiting tumor development in mice when administered i.p. 1 day after inoculation with sarcoma 180J ascites cells. Animals bearing solid tumors were not affected. In AKR/J mice with advanced leukemia, a single i.p. injection of 100 mug of the dimer led to about 50% reduction in the enlarged lymph nodes and the spleen at 24 hr. The half-life of the dimer in the bloodstream has been determined to be 10 min in rats and 6 min in mice, compared to values of 5 and 3.5 min, respectively, for the monomer. Analyses of the tissues of untreated leukemic mice for RNase and RNase inhibitors show that the tumor tissues are not deficient in RNase activity. Considerations of possible mechanisms of action of the dimer indicate that other basic proteins in this size range may merit examination as cytostatic agents toward transformed cells.


Prolactin levels in populations at risk for breast cancer.

The basal plasma concentration of prolactin was determined by radioimmunoassay in Caucasian women at different risk for breast cancer and in three cases of women at risk for breast cancer. Samples taken under comparable conditions showed similar basal levels at birth and in prepubertal Bantu and Caucasian girls, or in healthy pre- and postmenopausal Bantu, Caucasian, or Japanese women. Elevated plasma prolactin levels were found in women castrated prior to 35 years of age and in women whose first pregnancy occured after 35 years. Evidence indicates that the prolactin/estrogen relationship may not be similar in women 35 to 45 years of age as compared to young women and suggests that elevation of serum prolactin per se does not appear to be related to an increased risk of breast cancer. The prolactin level was increased only in Caucasian women with breast cancer. If a high prolactin/estradiol ratio increases the susceptibility of the mammary epithelium to neoplastic growth, the lack of changes in prolactin levels in premenopausal Japanese patients and in postmenopausal patients of the three ethnic groups indicates that other factors are involved. Further study of the effects of life-style and diet on the basal level and stimulated release of prolactin is required to resolve the relationship of prolactin to breast cancer.


Effect of N-methyl-N-nitrosourea on the DNA of rat bladder epithelium.

Bladder cancer can be induced in the rat by the intravesicular administration of N-methyl-N-nitrosourea. DNA damage in rat bladder epithelial cells after administration of methylnitrosourea has been examined by measuring the change in sedimentation of the DNA in alkaline sucrose gradients. A dose response of DNA damage in the urothelium was observed with single intravesicular doses of 0.1, 0.3, and 0.5 mg of methylnitrosourea. Larger doses of methylnitrosourea damaged the epithelium so extensively, that biochemical studies were not feasible. DNA repair, measured by the return to a normal sedimentation pattern of DNA on alkaline sucrose gradients, was followed over a period of 9 days with the use of 0.5 mg of methylnitrosourea to initiate the damage. Bladder epithelial cells were able to repair the DNA damage induced by methylnitrosourea. However, the possibility of persistent damage not detectable by sedimentation of DNA on alkaline sucrose gradients cannot be ruled out.


Immunological enhancement of leukemia L1210 by Corynebacterium parvum in allogeneic mice.

The effect of Corynebacterium parvum on the immune response of C57BL/6 mice (H-2b) to the allogeneic leukemia L1210 (H-2d) was investigated. Mice were either left untreated or given C. parvum i.v. or i.p. in various dosages. Seven days later they were challenged with 2.5 to 10 X 10(6) live L1210 cells i.p. Control animals almost always rejected the challenge. In contrast, most mice pretreated with either 1.0, 0.5, or 0.25 mg of C. parvum i.v. and 1.0 or 0.5 mg i.p. exhibited enhanced growth of leukemia L1210 as indicated by gross ascites and significantly greater weight gain. This sometimes progressed to the death of the animal, but more often regressed after several days. Spleen cell-mediated cytotoxicity to alloantigens, evaluated in vitro by release of 51Cr from P815Y (H-2d) target cells, was significantly decreased in the mice pretreated with either 1.0 or 0.5 mg of C. parvum i.v. or 0.5 mg of C. parvum i.p. This suppression could not be reversed by reduction of the concentration of macrophages in the spleen cell suspensions. Complement-dependent cytotoxic antibody, measured by release of 51Cr from L1210 cells, was profoundly suppressed in mice pretreated with C. parvum i.v. in dosages ranging from 1.0 to 0.1 mg. These data suggest an immunological basis for the enhanced growth of leukemia L1210 caused by C. parvum at these schedules.


Aryl hydrocarbon hydroxylase in mouse mammary gland.

The specific activity of aryl hydrocarbon hydroxylase in the mammary gland has been measured in six different mouse strains comprising both mammary cancer susceptibility and resistance, under basal conditions, and after pretreatment of the mice with 3-methylcholanthrene (3MC). No correlation was observed between the ease of 3MC-enhanced mammary tumorigenesis and the basal or induced specific activity of aryl hydrocarbon hydroxylase. Furthermore, it was shown that in the AKR/J and A+/Ki mice, only minor enhancement in aryl hydrocarbon hydroxylase was seen upon injection of 3MC. The C57BL/6J and C3Hf/He Tex mammary glands responded the most in this regard. It was also shown that the inability of the AKR/J or A+/Ki mice to respond was not due to their inability to absorb 3MC from the peritoneal cavity and transmit the polycyclic hydrocarbon to the mammary gland.


Quantitative studies of the toxicity of benzo(a)pyrene to a mouse liver epithelial cell strain in culture.

The toxic effects of the carcinogen benzo(a)pyrene (BaP) were studied in a well-characterized epithelial cell strain NMuLi, derived from the livers of weanling Namru mice. These cells were extremely susceptible to the toxicity, 99% dying after a 6-day exposure to BaP, 5mug/ml. The toxic effects began between 11 and 24 hr postapplication of BaP to the cells and increased exponentially with the time of treatment. The toxicity was concentration dependent in cells treated for a specific time period. The survival curves were exponential and extrapolated to a survival fraction of 1.0. The toxic effects of BaP to logarithmically growing NMuLi were inhibited 40% by 7,8 benzoflavone, and the inhibition was concentration dependent. The 7,8-benzoflavone also inhibited aryl hydrocarbon hydroxylase (AHH) from NMuLi cell homogenates and microsomes by 99%. The concentration dependence for AHH inhibition by 7,8-benzoflavone paralleled its inhibition of cellular toxicity. The toxicity of BaP to these cells increased exponentially with the number of population doublings. Hence, the toxicity was 130 times greater in exponentially growing cells than in confluent cells. Levels of AHH, the enzyme that metabolizes BaP to its cytotoxic derivatives, were only 2.4 times higher in exponentially growing than in confluent cells, suggesting that cell division was responsible for the large differential toxicity. In addition, a toxic BaP metabolite was preferentially toxic to log-phase cells. The results indicate that the metabolism of BaP by AHH to produce cytotoxic metabolites, which may cause lesions that are expressed upon cell division, is responsible for the cytotoxicity of BaP to NMuLi.


Induction of type B virions to bud into cytoplasmic vacuoles in a mammary tumor of an X-ray- and urethan-treated X/Gf mouse.

Electron microscopic studies of thin sections of a mammary carcinoma that developed in a female mouse of the syngeneic X/Gf low-cancer strain after treatments with X-rays (3 X 100 rads weekly) followed by 8 weekly i.p. injections of urethan (1 mg/g body weight) revealed extensive budding of B particles into cytoplasmic vacuoles and numerous characteristic B virions in the vacuolar lumens. Simultaneous budding of B particles into cytoplasmic vacuoles and budding of B particles at plasma membranes of the same cancer cell were also noted (figs. 2 to 6).


Differentiation of early mouse embryonic and teratocarcinoma cells in vitro: plasminogen activator production.

Cultured mouse blastocysts produce plasminogen activator, a protease that converts the zymogen plasminogen into the trypsin-like enzyme, plasmin. We have fractionated the blastocyst and cultured the constituent cell types. Trophoblast outgrowths free of inner cell mass derivatives secrete plasminogen activator during a time period that closely parallels the invasive phase of trophoblast cells in utero. Isolated inner cell masses also produce plasminogen activator; further fractionation of the inner cell mass as well as studies with primary cultures obtained from midgestation tissues demonstrate that enzyme formation is restricted entirely to parietal endoderm cells. Secretion of the enzyme may facilitate the migration of parietal endoderm cells along the trophoblast layer as the yolk sac cavity enlarges during gestation. F9 embryonal carcinoma cells do not secrete detectable amounts of plasminogen activator. However, when these cells are induced to differentiate, the resulting parietal endoderm-like cells are capable of producing the enzyme. These results are consistent with previous findings suggesting that plasminogen activator production may be a characteristic of invasive and/or migratory cells.


Evidence for altered gene regulation in HeLa cells returned to culture after growing in immunosuppressed rats.

HeLa TCRC-1, is a monophenotypic cell line for the production of the carcinoplacental Regan isoenzyme of alkaline phosphatase. It has been previously reported that, when this cell line is grown in immunosuppressed rats, there is an alteration of phenotypic expression in that an isoenzyme that is referred to as oncoamnion (FL) becomes the dominant enzyme form. This report describes the isoenzyme regulation after these cells are returned to culture for at least three months. These cells in culture demonstrate a density-dependent alteration in isoenzyme profiles. The Regan isoenzyme is the dominant isoenzyme form in new, sparsely populated cultures, while the oncoamnion isoenzyme predominates in the later high-density stages of growth. Prednisolone induction of enzyme activity is found to be most effective in the early stages of growth, and the previously reported specificity of hormone action on isoenzymes produced by cancer cells is maintained. Enzyme induction is restricted to the Regan isoenzyme, while hormone-mediated diminution in enzyme activity is confined to the oncoamnion (FL) isoenzyme.


Alkaline phosphatase isoenzymes in intestinal metaplasia and carcinoma of the stomach.

The enzymological and immunological properties of alkaline phosphatase (ALP) isoenzymes in intestinal metaplasia and gastric carcinoma tissues were compared with those of purified human intestinal, placental, and hepatic ALP's to speculate the gene expression of gastric cancer cells. The results were as follows: Intestinal metaplasia was found in 21 of 23 cases (91%) of gastric cancer, whereas normal gastric mucosa had no ALP activity. ALP extracted from the mucosa of intestinal metaplasia was identical with intestinal ALP as to enzymological and immunological properties. ALP obtained from gastric carcinoma tissues of 23 patients was separated into 3 bands (ALPa, ALPb, and ALPc) by polyacrylamide-gel disc electrophoresis. Ten cases showed ALPa only, 4 cases showed ALPa and ALPb, 5 cases showed ALPa and ALPc, and the other 4 cases showed ALPa, ALPb, ALPc, respectively. It was concluded that ALPa was similar in its enzymological and immunological properties to hepatic-type ALP, ALPb was similar to placental-type ALP, and ALPc was similar to intestinal-type ALP, ALPb probably originates from the cancer cell itself. As for the other enzymes, ALPa and ALPc, further investigation should be made to determine whether they come from the cancer cell itself or not.


A model for the control of transcription during development.

In order to account for the coordinate expression of large numbers of genes that must occur during development and differentiation, a mechanism of transcription control distinct from those operating in bacteria is proposed. Transcription would be initiated by small RNA chains that would function as primers for elongation. A given species of primer RNA generated by a primary induction event would bind by complementary base pairing to a variety of sites in the genome and thus trigger the transcription of adjacent structural genes. The primer RNA region would be excised from the transcription products in the nucleus, and possibly be reutilized as primers for transcription. This model can account for the occurrence of tissue-specific nuclear RNA complementary to repetitive DNA and does not require that the major RNA polymerases be capable of initiation. A minor RNA polymerase capable of initiating primer RNA chains and subject to conventional transcription controls would be required. Carcinogenesis, which is accompanied in many cases by the appearance of a variety of embryonic antigens, could involve the induction of RNA primers normally programmed to function early in development.


The role of nuclei and nucleoli in the control of cell proliferation.

A number of changes have been reported to occur in chromatin of quiescent cells stimulated to proliferate. These changes, indicative of increased transcriptional activity, occur in the early prereplicative phase, several hours before the onset of DNA replication, and are detectable not only in chromatin, but also in isolated nuclei and in whole cells. Most of the increased transcriptional activity can be attributed to an increased activity of the nucleolus; however, extranucleolar genes are also important in the regulation of the cell cycle flow, from G0 to S.


Role of nonhistone chromosomal proteins in the regulation of histone gene expression.

Histone gene expression was studied during the cell cycle of continuously dividing HeLa S3-cells and following stimulation of confluent monolayers of WI-38 human diploid fibroblasts to proliferate. The presence of histone messenger RNA (mRNA) sequences was assayed by hybridization to a 3H-labeled single-stranded DNA complementary to histone mRNA's. In HeLa S3-cells, histone mRNA sequences were found in the nucleus and associated with polyribosomes during S phase but not during G1. Transcripts of S-phase chromatin contained histone mRNA sequences but those of G1 chromatin did not. Similarly, in WI-38 cells association of histone mRNA sequences with polyribosomes and transcription of histone mRNA sequences from chromatin parallel DNA replication. Together these results suggest that the regulation of histone gene expression resides, at least in part, at the transcriptional level. Chromatin reconstitution studies provide evidence that nonhistone chromosomal proteins play a key role in activation of histone gene transcription during the period of the cell cycle when DNA is replicated. Phosphate groups associated with the S-phase nonhistone chromosomal proteins appear to be functionally involved in the control of histone gene readout. Although WI-38 human diploid fibroblasts transformed by SV40 exhibit morphological and biochemical modifications characteristic of neoplastic cells, transcription of histone mRNA sequences remains unaltered.


Fundamental carcinogenic processes and their implications for low dose risk assessment.

Various possible models of carcinogenesis are analyzed with respect to low dose kinetics. The importance of background carcinogenesis upon the shape of the dose-response curve at low dose is emphasized. It is shown that, if carcinogenesis by an external agent acts additively with any already ongoing process, then under almost any model the response will be linear at low dose. Measures of the degree of linearity are obtained for multistage models of carcinogenesis, where it is shown that throughout the dose range where the extra risk is less than the spontaneous risk linear extrapolation must be quite accurate.


Use of lipid vesicles as carriers to introduce actinomycin D into resistant tumor cells.

Unilamellar lipid vesicles have been used as a carrier vehicle to enhance the uptake of actinomycin D into an actinomycin D-resistant Chinese hamster tumor cell line (DC-3F/ADX). The DC-3F/ADX cell line is resistant to actinomycin D as a result of its decreased capacity to transport actinomycin D across the plasma membrane and is able to grow in the presence of concentrations of actinomycin D that are cytotoxic for the sensitive parent cell line (DC-3F). Incubation of resistant DC-3F/ADX cells with actinomycin D-containing vesicles produced a 5-fold increase in intracellular drug concentration over that achieved by exposure to identical concentrations of the drug added to the culture medium. Vesicle-mediated uptake of actinomycin D into resistant cells produced inhibition of cellular RNA synthesis and cell growth at actinomycin D concentrations that had no inhibitory effect when added as free drug in the culture medium. Dose-response measurements established that direct introduction of actinomycin D into resistant cells by means of lipid vesicles resulted in a 200-fold reduction in the concentration of actinomycin D required to inhibit cellular RNA synthesis and a 120-fold reduction in the dose of actinomycin D required to produce a 50% inhibition of cell growth. These results lend strong support to the hypothesis that cellular resistant to actinomycin D is due to a lower capacity to take up actinomycin D as a result of a reduction in the permeability of the cellular plasma membrane to this drug. The potential value of lipid vesicles for introducing other classes of drugs into cultured cells and their possible use in chemotherapy are also discussed.


A rapid assay for stimulation of human lymphocytes by tumor-associated antigens.

Lymphocyte-stimulated protein synthesis (SPS) in response to human tumor-associated antigens was assessed by measuring [3H]leucine incorporation. Correlation of SPS with other in vivo and in vitro response was demonstrated by immunizing normal subjects with keyhole limpet hemocyanin and testing sequentially frozen lymphocytes and serum samples. One week after immunization, lymphocytes from normal subjects demonstrated increased SPS to keyhole limpet hemocyanin. This correlated with the appearance of delayed cutaneous hypersensitivity responses and preceded detection of hemagglutinating antibodies and increases in lymphocyte [3H]thymidine incorporation. There was no difference in the reactivity of fresh and viable frozen lymphocytes, and as few as 5X 10(5) lymphocytes/microtiter plate well could be used. Tumor-associated antigens were prepared from four lung carcinomas, six sarcomas, and six melanomas, using 3 M KCI extraction. Lymphocyte responses to both autologous and allogeneic tumor extracts were observed. Five of 15 patients demonstrated significant SPS to autologous tumor antigens. Fourteen of 20 lung cancer patients responded to lung cancer antigen, whereas only 11 of 41 patients with other tumors and 3 of 19 normal subjects reacted. Significantly, more lung cancer patients reacted to the tumor extract than to an extract of uninvolved lung from the same patient. Twenty-one of 42 melanoma patients responded to melanoma antigen. Ten of 33 patients with other tumors and 3 of 24 normal subjects reacted to the melanoma extract. Eight of 30 melanoma patients reacted to an extract of muscle from the same donor as was the melanoma antigen. Tumor-associated antigenic activity of 3 M KCI extracts can therefore be detected by measuring lymphocyte [3h]leucine incorporation.


Effects of single cyclophosphamide doses on the kinetics of thoracic duct lymph and blood leukocytes in calves.

After 5 or 20 mg cyclophosphamide per kg body weight, given once i.v., the output in thoracic duct lymph of small and large lymphoid cells, cells incorporating [3H]lymidine in vitro, mitotic cells, pyknotic cells, and/or the number of lymphocytes and neutrophils in peripheral blood were measured in six calves. The median grain count of labeled cells and the DNA content of pyknotic nucleic were determined. After both doses there was an exponential decrease and subsequent recovery of the median grain count. The larger dose caused a temporary cessation of lymphoid cell division, reduced the output of nondividing small lymphoid cells, and probably imparied proliferation of neutrophil precursors. The results suggest that increased cell production during recovery was due to changes in the growth fraction and that feedback mechanisms acting on G0-G1 cells controled the proliferation of lymphoid cells.


Kinetics of the plasmacellular reaction in the draining lymph node of mice bearing the BP-8 fibrosarcoma.

The kinetics of the plasmacellular response in the draining lymph node was quantitatively assessed in specific-pathogen-free C3H/He mice grafted S.C. with the syngeneic BP-8 fibrosarcoma. The percentage of plasma cells was estimated on dissociated, cytocentrifuged, and fixed cell preparations by immunoenzymatic staining with either of two B-cell-specific antibody markers. Mice either grafted with liver tumor or given a single implant of tumor homogenate gave a maximal plasma cell response of about 4%. However, this peak was reached at 14 days in the live grafts and at 7 days in the homogenate situations. Moreover, in animals grafted with live tumor an elevated 3% plateau persisted until death, wheras in homogenate-implanted animals a sharp drop to a 0.5% plateau was observed at about Day 20. Homogenate-treated animals displayed a significant enhancement of tumor growth when challenged with a live graft 7 days after homogenate implantation, i.e., during the peak 4% plasmacellular response. Mice given a single implant of X-irradiated (3500 R) tumor responded with a peak of 1.7% plasma cells at 7 days. Despite subsequent boosting with the same material (Days 14 and 21), this value declined and remained at a plateau of about 0.2% until death. Contrary to homogenate-treated mice, these animals were immune to subsequent challenge with a live tumor graft. In the present host-tumor system, there appears to be an inverse relationship between elevated numbers of plasma cells and antitumor immunity.


Increased attachment of nucleic acids to eukaryotic and prokaryotic cells induced by chemical and physical carcinogens and mutagens.

Significantly enhanced attachment to Ehrlich ascites and Escherichia coli cells was observed for radioactive DNA and RNA in the presence of chemical mutagens and ultimate carcinogens. In some instances, formation of nucleic acid-protein adducts by these compounds further (or similarly) enhanced the binding. DNA irradiated with ultraviolet light in the presence of a protein bound more efficiently than either an unirradiated mixture of these two macromolecules or DNA irradiated alone. The spectrum of compounds tested and found active in this system includes alkylating agents, aromatic amines, and carcinogenic metals. Precarcinogens and nonultimate carcinogenic chemicals, as well as tumor-promoting agents, did not increase the binding. However, addition of extracts from mouse or rat livers activated precarcinogenic and proximate carcinogenic chemicals  and resulted in enhanced cellular attachment of indicator nucleic acids in their presence. Possible usefulness of this test system for fast and efficient screening for environmental carcinogens and mutagens, as well as possible relevance of the observed phenomena to in vivo effects of chemical and physical carcinogens, is considered.


Retention of tritiated methotrexate in a transplantable mouse glioma.

The uptake and distribution of tritiated methotrexate ([H]MTX) were studied by autoradiography and liquid scintillation countinf for up to 7 days after i.v. injection in mice bearing s.c. or intracerebral (i.c.) implants of a transplantable mouse ependymoblastoma. Autoradiography showed that s.c. tumors took up more [3H]MTX than i.c. tumors. In both s.c. and i.c. tumors, [3H]MTX was mainly intracellular, with very little in intersitial fluid sites. Retention of [3H]MTX gradually diminished with time, with some still being present 7 days after injection. The distribution of [3H]MTX in the i.c. tumors was not uniform, and portions of these tumors were relatively inaccessible to the drug. The uniformity of distribution did not improve with time. Scintillation counting showed that s.c. tumors accumulated much less [3H-A1MTX and retained a lower proportion of [3H]MTX than many normal tissues. These studies indicate that [3H]MTX has 3 major shortcomings as a chemotherapeutic agent for brain tumors. First, the amount of drug taken up by the transplantable mouse ependymoblastoma was small in comparison with normal tissues and in comparison with other agents taken up by this tumor. Second, the distribution of the drug in the i.c. tumors was nonuniform, with tumor cells in certain areas remaining relatively inaccessible to the drug. Last, the retention of the drug in the tumor was far less than in normal tissues.


Antitumor activity of 9-hydroxyellipticine (NSC 210717) ON L1210 mouse leukemia and the effect of route of injection.

The antitumor activity of a new derivative of ellipticine, 9-hydroxyellipticine (NSC 210717), was studied using L1210 mouse leukemia. Low doses of this drug have a high antileukemic activity, whereas high doses have less activity than expected because of a leveling off in the antitumor activity-dose relationship, as if a few cells were resistant to the treatment. The possible causes of this apparent resistance were investigated. It is suggested that this apparent resistance is related to the sequestration of a small number of cells in compartments inaccessible to the drug. A model was developed which takes into account the distribution of cells in various compartments and their drug sensitivity therein. It was predicted and observed that the activity of drugs acting on cells in the small compartments can be observed only in conjunction with the presence of drugs acting on the cells in the major compartment. The importance of this observation in the screening procedures of new drugs, the clinical trial of new chemotherapeutic agents, and the association of anticancer drugs are discussed within this context. 9-Hydroxyellipticine is of interest because it acts on leukemic cells in the brain.


Combined adriamycin and hyperthermia treatment of a murine mammary carcinoma in vivo.

A study was made of the effect of combined adriamycin and hyperthermic treatment in a solid mouse mammary carcinoma in vivo. This study demonstrated: (a) that, when given separately, adriamycin and hyperthermia enhance the destruction of a solid mouse mammary carcinoma in vivo; hyperthermia (40.5-42.5 degreesy greatly increases tumor destruction and, in a number of cases, caused initial and long-time regression; (c) that whole-body hyperthermia in combination with adriamycin gives a significant delay in tumor growth as compared with the controls, but not to the same degree as the local combined therapy; and (d) that treatment with local hyperthermia and adriamycin gives a pronounced decrease in the lethal toxicitity of adriamycin. The effect of adriamycin and heat treatment may be due to hyperthermic cell destruction in the central area of the solid tumor, together with a synergistic effect of heat and adriamycin on the proliferating peripheral tumor cells. Furthermore, local heat application may increase the adriamycin concentration in the heated tumor area, which causes a high destructive effect and a less toxic influence on the nonheated normal tissue.


Ultrastruct and biochemical chantes in cultured human malignant melanoma cells after heterotransplantation into nude mice.

Cells from three lines of cultured human malignant melanomas were heterotransplanted into nude mice and then recultered. The shape of the cells, the aspect of the melanosomes, and the content of 5-S-cyteinyldopa showed pronounced changes induced by the transplantation. Such results indicate that this experimental model should be used with great caution. A relationship was found between the shape of the melanosomes and the content of 5-S-cysteinyldopa in the cells.


Transport of anionic substrates and glutamate metabolism in mitochondria from ascites tumor cells.

A study is presented of alpha-oxoglutarate and glutamate transport and of glutamate oxidation in ascites tumor cell mitochondria. Kinetics analysis of alpha-oxoglutarate transport in mitochondria from two strains of Ehrlich ascites tumor cells, the hyperdiploid and the hyperdiploid Lettré mutant, shows that the activity of the alpha-oxoglutarate carrier and its affinityfor substrates are higher in the mutant than in the wild strain. Evidence is presented showing the occurrence of carrier mediated glutamate-OH-exchange-diffusion in mitochondria from both strains. The activity of the glutamate carrier is apparently higher in the hyperdiploid Lettré mutant. Glutamate oxidation occurs mainly through transamination to asparatate in both tumor stains. The rate of deamination in the two strains correlates directly with the level of glutamide dehydrogenase (EC 1.4.1.3.), which is higher in the wild than in the mutant strain. Thus glutamate dehydrogenase per se, and not glutamate penetration, constitutes the control step for gluttochondria of glutamate with externally added oxaloacetate (arsenite present) that exclude an obligatory transport of oxaloacetate on the alpha-oxoglutarate carrier.


Cell cycle and morphological changes during growth and differentiation of a rat basophilic leukemia cell line.

Cells of the rat basophilic leukemia cell line RBL-1 differentiated maximally when permitted to achieve growth arrest in a high-density stationary phase, in which the cell number is constant, and the cells are arrested in a G phase of the cycle. Features of differentiation are the accumulation of large basophilic granules and increases in membrane receptors for immunoglobulin E. However, changes in histamine content did not parallel granule development or changes in immunoglobulin receptor concentration. During rapid "forced exponential" growth, the cell number doubles every 8 hr, 50% of the cells are in S phase, and differentiation is minimal.


A comparison of the loosely bound and tightly bound nonhistone proteins from Ehrlich ascites tumor chromatin.

The nonhistone proteins of Ehrlich ascites tumor chromatin have been separated into a loosely bound and two tightly bound protein fractions by sequential extraction of chromatin with 0.35 M NaCl and 2 M NaCl:5 M urea. The nonhistone proteins thus obtained were examined for their chemical composition and distribution of DNA polymerase, RNA polymerase, and protein kinase activities. In addition, the effect of these nonhistone proteins on transcription of DNA in vitro has been determined. The results indicate that these nonhistone proteins, fractionated on the basis of their extractability, exhibit varied compositional characteristics and play different functional roles in the synthesis of DNA and RNA and in the possible control of gene activity.


Effects of the immunosuppressive drug niridazole in isogeneic and allogeneic mouse tumor systems in vivo.

In both isogeneic (Sarcoma 1 in A/JAX mice) and allogeneic (Sarcoma 180 in C57BL/6 mice) mouse tumor systems, treatment of the tumor-bearing mice with niridazole, an antiprarasitic drug, known to be a potent suppressor of cell mediated but not humoral immunity caused enhancement of metastases to regional popliteal nodes. Niridazole also inhibited tumor growth in vivo, as manifested by a significant decrease in the weight of the primary tumors. The enhancement of metastases is attributed to the suppression of cell-mediated immunity by the drug, but the mechanism of tumor-growth inhibition is not yet clear.


Preferential inhibition of DNA synthesis in mouse hemopoietic cells by halothane.

The effect of prolonged light halothane anesthesia (0.8%) on the proliferation rate of different mouse tissues was investigated, using [5-125I]5-iodo-2-deoxyuridine uptake into DNA as the test parameter. It was found that DNA synthesis in spleen, femoral bone marrow, and, occasionally, the small intestine was significantly depressed after exposure for 24 hr to halothane in vivo. The time course of DNA synthesis inhibition was then investigated by utilizing a shorter (6-hr) exposure time. This period was found to be insufficient to cause DNA synthesis inhibition in any of test tissues. Because anesthesia was found to be associated with hypothermia at normal room temperatures, it was established that the inhibition of DNA synthesis was not due to cooling of the mice under anesthesia by demonstrating that inhibition in sensitive tissues occurred at warmer temperatures as well. To examine the specificity of this finding, the DNA synthesis rate of cells in other normal tissues, e.g., skin and muscle, and in s.c.-growing tumor cells of a mouse mammary carcinoma, L1210 leukemia, and a first transplant AKR lymphoma were examined. In none were responses noted with 24 hr of halothane exposure. However, halothane was found to inhibit DNA synthesis in regenerating marrow. Finally, it was found that after significant exposure to halothane, complete recovery was seen in the spleen after 24 hr, whereas femur DNA synthesis was still depressed by 20% at the same time.


Characterization in vivo and in vitro of tumor cells selected for resistance to syngeneic lymphocyte-mediated cytotoxicity.

This report describes the selection and behavior of tumor cells resistant to cytolysis by syngeneic lymphocytes. Two B16 melanoma lines, F1 (low metastasis) and F10 (high metastasis), were cultured with lymphocytes from C57BL/6 mice immunized against B16. The  selection procedure involved repeated exposure of the tumor cells to lymphocytes in vitro. After each interaction, the viable tumor cells were trpsinized, replated, and designated lines F1Lr-1 and F10Lr-1. The procedure was repeated five times, yielding lines F1Lr-6 and F10Lr-6, which resisted cytolysis by syngeneic lymphocytes. Mice were given s.c. or i.v. injections of cells from lines F1, F1Lr-6, F10, or F10Lr-6. Tumor growth patterns were the same for all four lines when the cells were injected s.c., however, the incidence of pulmonary metastases differed significantly after i.v. injection. Line F10 cells yielded more pulmonary metastases than an equal number of line F1 cells (p less than 0.01). F1Lr-6 cells yielded significantly fewer metastases than an equal number of lines F1 cells (p less than 0.01). A similar difference between F10Lr-6 and F10 cells was observed. The incidence of artificial metastases after i.v. injection of F10Lr-6 cells was similar to that for F1 cells. The quantitative organ distribution, arrest, and survival of i.v.-injected tumor cells were studied by using [125]-5-iodo-2'-deoxyuridinelabeled cells. There was a significantly greater number of cells from line F10, arrested and able to survive for 14 days in lungs, than cells from line F1. In contrast, cells from either line F1Lr-6 or F10Lr-6 had a lower incidence of arrest and survival than their lymphocyte-sensitive counterparts.


Effects of Dintrato(1,2-diaminocyclohexane)platinum (NSC 239851) On murine myeloma and hemopoietic precursor cells.

We studied the effects of dinitrato (1,2,-diaminocyclohexane)platinum (NSC 239851) on murine myeloma  Adj. PC-5 and hemopoietic precursor cells. The median survival time of tumor-bearing mice was significantly affected by i.p. injections of this agent. While 14 mg/kg was a toxic dose, as little as 2 mg/kg prolonged the survival for over 100 days. When cells from normal marrow and actively regenerating marrows were exposed to the agent in culture tubes, washed, and assayed for the surviving hemopoietic precursors, similar sensitivity curves were observed. This result indicates the absence of cell cycle dependency of the agent. When marrow cells were exposed to NSC 239851 at 4 degrees in culture, almost total abrogation of the cytotoxicity was noted. This observation, unlike that from the experiment with the inorganic platinum congener, cis-diamminedichloroplatinum, suggests that the transport of this agents is an active process.


Homocysteine thiolactone metabolism in malignant cells.

Since abnormal homocysteine metabolism is associated with several disorders of growth, including neoplasia, the metabolic fate of homocysteine thiolactone was studied in cell cultures from several malignant tumors, established cell lines, cell lines transformed by oncogenic viruses, and normal skin cells. In all of the cultures of malignant cells homocysteine thiolactone becaome incorporated in peptide linkage with cellular proteins (thiolation). Normal cells incorporated homocysteine thiolactone in peptide linkage, but homocysteine thiolactone was released by acid hydrolysis. The findings suggest the speculative possibility that malignant cells are deficient in a homocysteine thiolactone derivative that prevents thiolation of proteins by homocysteine thiolactone. This hypothetical substance may also catalyze the synthesis of methionine and release acrolein, a growth-regulatory substance, in normal cells. The growth characteristics and tumorigenicity of cultured cells may be related to depletion of the hypothetical substance, and its identification, synthesis, and administration to animals would be expected to affect growth of malignant neoplasms.


The effect of adriamycin and radiation on G2 progression.

The effect of the DNA-intercalating antibiotic adriamycin on the progression of Chinese hamster ovary cells into mitosis, and on the delay induced by ionizing radiation, was studied using the mitotic cell selection procedure to monitor the rate of cell division. Following the addition of adriamycin, the mitotic rate remained unaltered for a refractory period and then decreased to zero. This effect was concentration dependent with transition points between the S-G2 boundary for 0.1 mug/ml and late G2 for 250 mug/ml. Cells treated with either a 10- or 30-min pulse of 1.0 mug adriamycin per ml exhibited a refractory period identical to that observed for continuous treatment. However, after a delay of congruent to 3.5 or congruent to 5 hr, respectively, cell division resumed. The mitotic rate of cells that received 150 rads of X-ray at the oneset of an adriamycin pulse declined coincident with that of radiation only, but resumed coincident with those receiving adriamycin only. This implies that radiation-induced division delay (congruent to 3 hr) was repaired before cells recovered from adriamycin-induced division delay and that the two agents were not additive. This lack of synergism is in contrast to that observed for cell lethality.


Decreased prevalence of immediate hypersensitivity (atopy) in a cancer population.

It has been suggested that the atopic population has decreased risk of cancer. This investigation examined the cumulative prevalence of atopy in a population with neoplastic disease and compared this with the prevalence of atopy in an age-matched control group and with published estimates of atopy in the general peopulation. Seventy-four patients with neoplastic disease and 86 patients without cancer were evaluated. The subjects were given a standard allergic questionnaire which evaluated them with regard to a history of allergic symptoms, hives, eczema, frequent colds, frequent unexplained rashes, hay fever, and asthma. All were skin tested with a representative group of regionally significant allergens. There was a 15-fold decrease in prevalence of atopy in the cancer population, compared with the control group and compared with published estimates of atopy in the general population.


Identification and characterization of a circulating tumor-associated oncofetal protein from a radiation-induced adenocarcinoma of the rat small bowel.

A tumor-associated protein from the cellular membranes of a radiation-induced rat small bowel adenocarcinoma was identified, found to be serologically unaltered in the circulatory system, and was observed to be susceptible to acid hydrolysis. The immunochemical reactivity was unchanged by heat, alkali, or neuraminidase digestion. The protein appeared to be a single immunologically active species, but it was structurally composed of a heterogeneous group of proteins.


Growth stimulation of cultured endothelial cells by tumor cell homogenates.

Short-term cultures of endothelial cells from fetal bovine heart and aorta consistently display increased growth rates when crude tumor cell homogenates from the Walker 256 adenocarcinoma (ascites and solid forms as well as tumor cells from a suspension culture) are added to the culture media. The tumor-derived material produces growth stimulation in both sparse and confluient endothelial cell cultures. Homogenates of embryonic tissues and cultured cells show similar growth-promoting effects; corresponding material from various adult tissues is ineffective. The nonendothelial cells that were tested generally show no growth response or only a slight positive growth response to the tumor cell homogenates. The results indicate the feasibility of using this in vitro system as a paradigm of the tumor-induced vascularization process.


A rapid in vitro labeling index method for predicting response of human solid tumors to chemotherapy.

A rapid autoradiographic technique for measuring the [3H]thymidine-labeling index of human solid tumors has been adapted to assess the effect of anticancer drugs in vitro. The drugs tested were present unchanged or as metabolites in serum obtained from patients immediately post-treatment. In 15 patients, the drugs tested in vitro were also given in vivo. Tumor-labeling index fell significantly in 5 of 6 patients who were later found to have objective clinical response. Tumor-labeling index did not change significantly in 8 patients and rose significantly in 1 of 9 patients who lacked clinical response. If confirmed, this in vitro test may prove to be a useful method of predicting responsiveness of human solid cancers to chemotherapeutic agents.


Effects of 5-mercapto-2'-deoxyuridine on the incorporation of nucleosides into RNA and DNA in a primary lymphocyte culture system.

The effects of 5-mercapto-2'-deoxyuridine (MUdr) on DNA synthesis in a primary murine spleen lymphocyte culture system stimulated by phytohemagglutinin (PHA) were studied. Inhibition of thymidine incorporation into acid insoluble nucleic acid material was 50% at 0.5 mM MUdR concentration, while inhibition of deoxyuridine incorporation into acid-insoluble nucleic acids was 50% at 0.01 mM MUdR. Time course studies, at 0.5 and 0.05 mM MUdR, showed that the magnitude of inhibition of incorporation for thymidine and deoxyuridine, respectively, increased from a time point after PHA stimulation when increased synthesis of thymidine kinase and thymidylate synthetase had leveled off. At 1 mM MUdR, total cellular DNA in cultures was decreased 43% at 42 hr after PHA stimulation. Neither the total number of cells nor the percentage of PHA-transformed cells was decreased in comparison to that of controls. MUdR therefore blocks the increase in DNA content of lymphocytes that is initiated during the S phase of the cell cycle. Millimolar levels of MUdR inhibited incorporation or uridine, adenosine, and cytidine into acid-insoluble material in pha-stimulated primary murine lymphocyte cultures. Total cellular RNA synthesis was inhibited at these levels of MUdR, with no differential effects on 4, 18, or 28 S RNA species observed. Uptake of these nucleosides into the total cellular acid-soluble material was not blocked. Uptake of different labeled nucleosides into cellular, acid-soluble pools occurs at different rates. Thus, choice of a suitable minimum pulse time to achieve saturation for different labeled nucleosides must relate to this consideration. Thymidine kinase from whole-cell sonic extracts of PHA-stimulated lymphocytes was inhibited 65% by 1 mM MUdR at 24 and 48 hr after stimulation. Uridine kinase extracted from the PHA-stimulated cells was also significantly inhibited by 1mM MUdR at 24 hr (56%). Exogenous guanosine incorporation into lympohcyte acid-insoluble material is increased by MUdR. This increased utilization of exogenous nuceloside is apparently the result of MUdR inhibition of conversion of adenosine to guanine nucleotides within the lymphocytes and a consequent diminution of the total intracellular guanine nucleotide pool size. The active inhibitory compound is the deoxyribonucleoside or deoxyribonucleotide. Comparison with the riboside analog 5-mercaptouridine showed that MUdR was a more efficient inhibitor of nucleoside incorporation.


Relationship of cell growth behavior in vitro to tumorigenicity in athymic nude mice.

The serum requirements, anchorage requirements, saturation densities, and contact inhibition responses of a variety of mammalian cell lines were determined under uniform conditions. The serum requirement of both transformed and normal cells was a sensitive function of initial plating density. Cloning efficiency on irradiated mouse monolayers was found to be an invalid indicator of contact inhibition of growth, since most cell lines that failed to form visible colonies on cell monolayers nonetheless proliferated on these monolayers. When normal and neoplastic cells from a variety of sources were examined, none of the growth parameters that serve to define the transformed state in vitro correlated consistently with cellular tumorigenicity in athymic nude mice. It is concluded that the most reliable and physiologically meaningful assay for malignant transformation is, at present, cellular tumorigenicity in athymic nude mice.


Changes in ornithine decarboxylase activity in rat intestines during aging.

The ornithine decarboxylase (ODC) activity of rat intestines, liver, and brain was found to vary dramatically as animals develop and age. Unusually high activity was present in the small intestines of adult animals. The ODC activity of the small intestines approximated that of fetal tissues and of regenerating rat liver. Putrescine, spermine, and spermidine levels of fetal and adult animal tissues were determined. In all tissues but the stomach mucosa, high putrescine levels correlated with high IDC activity. However, the total polyamine concentrations of the stomach and colon could not be correlated with ODC levels, and no simple relationship between polyamine levels of ODC levels and cellular proliferation in the gut was found.


Detection of occult metastatic melanoma by urine chromatography.

By using ion-exchange column chromatography with effluent monitoring using the stable, free radical alpha,alpha-diphenyl-beta-picryhydrazyl as a colorimetric reagent, we have demonstrated the occurrence of elevated levels of five peaks in the urine of patients with metastatic disease. The tentative assignment of two of the peaks as 3,4-dihydroxyphenylalanine and as 3-methoxy-4-hydroxyphenylalanine has been made. Three remain unknown. The correlation of these peaks with the clinical status of melanoma patients shows that, while the individual excretion pattern of these compounds may be variable, the sustained occurrence of one or more of them in a patient's urine is evidence of recurrent or continuing disease. The excretion levels appear to be proportional to the tumor burden. The results with a group of 39 melanoma patientshaving Stage II or Stage III disease indicate that this chromatography technique provides earlier evidenc eof liver metastases than doses the liver scan, may detect occult metastases generally, and has detected tumor in clinically enlarged lymph nodes. This method, in its present form, does not detect small pulmonary lesions earlier than chest X-ray or tomography do or brain metastases earlier than do brain scan or computerized axial tomography. The technique is clinically useful for the diagnosis of melanoma patients and in their follow-up while under treatment.


Transfer RNA species in tumors of different growth rates.

Tyrosyl-, histidyl-, lysyl-, and phenylalanyl-tRNA's from 3 tumors (DBAH, DBAH, and DBA), differing in growth rates and from host mammary glands and liver, were compared by means of methylated albumin kieselguhr (MAK) column and by reverse-phase-5 chromatography. The elution profiles of lysyl-tRNA's from DBAH and DBA, phenylalanyl Trna's from DBAH and DBAH, and histidyl-tRNA's from DBA3 tumors exhibited extra isoaccepting species, compared with host liver and mammary glands. The distribution of acylatable tyrosyl-tRNA's in DBA3 and DBAH, phenylalanyl-tRNA's in DBAH and DBAH2, and histid-l-tRNA's in DBA3 is higher than that in liver, whereas no appreciable differences were observed in the lysyl-tRNA contents of the tumors and liver. The chromatographic alternations appeared to be a property of the tumor tRNA's and not due to differences in aminoacyl-tRNA synthetases or due to the aggregation of tRNA's. The structural and functional significance of these findings are discussed.


Characterization of dextran sulfate-treated ascites tumor cells and their repair by ascites fluid.

Exposure of Ehrlich ascites tumor cells to the polyanion dextran sulfate altered the permeability of the plasma membrane resulting in decreased glycolysis and respiration, enhanced permeability to Rb+ and adenosine 5'-triphosphate, and increased staining by erythrosin B. The inhibition of respiration was reversed by the addition of inorganic phosphate, whereas restoration of glycolysis required both inorganic phosphate and adenosine 5'-monophosphate. The membrane lesion was repaired by injection of dextran sulfate-treated cells into mice or by in vitro exposure to a heat-stable extract from ascites fluid. Reparied cells were no longer stained by erythrosin B and glycolyzed without added adenosine 5'-monophosphate. The time course of repaire was separable into a rapid initial repair of the membrane lesion followed by a slower recovery of the normal intracellular ionic composition. Both phases were insensitive to inhibitors of protein synthesis. The repair activity of ascites fluid was heat stable and was composed of both dialyzable and nondialyzable factors. Incubation of dextran sulfate-treated cells with ascites fluid restored Rb+ uptake and abolished the adenosine 5'-triphosphate-stimulated Ca2+ uptake.


Effect of deoxyribonuclease on adriamycin-polynucleotide complexes.

Recent interest in the use of adriamycin-DNA complex as an approach to improve the therapeutic effectiveness and to reduce toxicity of adriamycin for cancer chemotherapy requires an in-depth understanding of the physicochemical and biochemical properties of such complexes. The interactions of adriamycin with single-strand polydeoxyribonucleotides, double-strand DNA, and double-strand ribodeoxyribopolynucleotide hybrids were therfore investigated. Association constants (Kapp) of adriamycin and polynucleotides were obtained. These data showed that the inherent variable in such complex lies in the composition of the polynucleotides. Alternate deoxyguanylate (dG)-deoxycytidylate (dC) sequence binds 7-fold better than alternate deoxyadenylate (dA)-deoxythymidylate (dT) sequence. Comparative studies of the hydrolysis of DNA duplexes by deoxyribonucleases I and II with and without adriamycin were also carried out. The rate of hydrolysis decreased in the order poly(dA-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase I and poly(dA)-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase II. Intercalation of adriamycin to deoxyribopolynucleotide duplex resulted in inhibition of DNase II two to three times more than tat of DNase I. On the other hand, intercalation of adriamycin to homodeoxypolynucleotide duplex poly(dA)-poly(dT) and poly(dG)-poly(dC) enhanced the DNase I hydrolysis. If DNase I activity could be related to serum DNase and DNase II related to tumor lyososomal DNase as in the endocytosis mechanism proposed by Trouet et al. (Cancer Chemotherapy Rept., 59: 260, 1975), the best adriamycin carrier suggested by this investigation could be poly(dA)-poly(dT) and poly(dG-dC). It is also suggested in this study that adriamycin-RNA-DNA hybrid could be of interest as an antiviral agent by a similar release mechanism via RNase H, an enzyme associated with viral reverse transcriptase.


Potential regulatory roles for RNA in cellular development.

In addition to its widely accepted messenger and structural roles, RNA has been implicated in several other biological events: transfer of specificity of the immune response, interferon induction, and infection of plants by viroids. In none of these cases is the mechanism of RNA action understood. As a result of recent advances in the understanding of RNA metabolism in eukaryotic nuclei and potential specificity of nucleases that cleave such RNA, we have suggested specific ways in which "extra" RNA could be involved in regulation of the development of cells. A survey of literature concerning mammalian cell differentiation both in embryos and in the immune system leads to the observation that there appears to be a line of communication between macromolecules on cell surfaces and the genome. Making the conservative assumption that the DNA retains its integrity throughout development, it seems likely that highly specific signals are sometimes required to transfer information from outside the cell to the DNA,resulting in a change in state of differentiation. We propose a way in which RNA could be utilized in this process.


Selective control of DNA helix openings during gene regulation.

DNA helices must undergo openings in the form of localized strand separations in order to permit the onset of RNA synthesis or DNA synthesis. The selective control of such DNA helix openings at particular gene loci is the critical feature of gene regulation in prokaryotes and eukaryotes.


Continuity of a rabbit antigen between generations.

The rabbit uterine protein, blastokinin, is detectable as an antigen in both male and female genital tract tissues, in preimplantation embryos, and in lungs from late fetal stages, immatures, and adults. Thus, it serves as an example of how an antigen of normal tissue exists in a developmental progression from one generation to the next.


Activation of developmental genes in neoplastic transformation.

The view is advanced that it is important to fix the stage of development corresponding to the pattern of expression of oncodevelopmental proteins in neoplastic transformation and neoplasia. From a map of development focused on events beginning with the zygote and ending with the establishment of fetus and placenta, it is possible to explain why certain developmental gene products are restricted to fetus or placenta or distributed in both. It also suggests which products can be expected to be expressed concordantly in neoplastic transformation and in neoplasia.


Fetal growth modified by natural and induced changes in maternal reactivity.

Maternal immunological unresponsiveness has usually been demonstrated with adult tissue allografts. Experiments were first directed to the question of whether maternal reactivity to allogeneic conceptuses parallels that of implants of allogeneic tumor. BALB/c female mice parous by C3H males for one to eight litters and challenged with Tumor 70429 of C3h origin showed increasing unresponsiveness with multiparity. A high percentage of progressively growing tumors in highly multiparous females indicates that unresponsiveness does not give way to sensitization. The effects of multiparity on placental and fetal weights in BALB/c females pregnant by C3H males resulted in a continuing decrease in placental weight (indicative of unresponsiveness) through the third pregnancy, but this was followed by a contrasting progressive weight increase indicating sensitization with four or more pregnancises. Syngeneic placentas displayed a similar pattern suggesting that weight changes were not entirely alloantigen dependent and that fetus-specific antigens also alter maternal reactivity. The effects of specific and nonspecific maternal preimmunization and specific parity on placental weights were tested as follows. One group of BALB/c females was immunized against C3H (specific), a second was immunized against DBA/2 (nonspecific), and a third was not immunized. Each of these groups was divided in half and mated to either C3H or DBA/2 males for a first litter; then all were mated to C3H for a second pregnancy. Placental weights were significantly smaller in females that had their first litter by C3H males. A similar experiment with BALB/c X C57BL F1 females resulted in fewer changes in placental weights. In all instances, maternal reactivity had its major effect upon placental rather than fetal weight. The effect of maternal unresponsiveness upon reproductive capacity was tested by mating BALB/c females to either DBA/2 POR C3H males for a first litter and then mating all females ot C3H males for a second litter. Second litters were significantly larger when first litters were also sired by C3H males.


The significance of embryonic reexpression in cancer.

Irradiated syngeneic mouse testicular cells and the first-trimester human fetal tissue cells protect mice partially against tumors induced by methylcholanthrene or against a syngeneic transplantable mouse tumor, Meth A, respectively. For future research it is suggested that experiments should have a therapeutic rather than a prophylactic design and that the induction of tolerance to fetal antigens could be a highly informative procedure.


Antigens associated with normal and malignant gastrointestinal tissues.

Immunization of hamsters with phenol-alcohol extracts of GW-39 human colonic tumor tissues has resulted in the identification of three gastrointestinal tissue-associated antigens, on the basis of precipitin immunoreactivity. Sephadex G-200 and Bio-Gel A-15m chromatography of normal colonic tissue and GW-39 tumor extracts revealed antigen immunoreactivity in the 46,000 (low-molecular-weight), 170,000 to 900,000 (high-molecular-weight), and 5 to 10 million (very high-molecular-weight) ranges or low-molecular-weight colon-specific antigen (LMW/CSA), high-molecular-weight colon-specific antigen (HMW/CSA), and very-high-molecular-weight colon-specific antigen (VHMW/CSA), respectively. Immunodifussion reactions indicated that the HMW/CSA was human gastrointestinal tissue-specific, increasing in concentration from the esophagus to the colon [for which reason the term colon-specific antigen (CSA) has been retained], whereas the LMW/CSA was found in human gastrointestinal tissues, hamster and rat colon, human saliva, and normal human cervix. Colon-specific antigen (CSA) could be demonstrated in human gastrointestinal tumors, including the LS-174T colonic cancer cell line, but not in cancers of other sites tested. Likewise, CSA's were found in fetal human gut tissue. Whereas HMW/CSA and VHMW/CSA showed partial identity in immunodiffusion, HMW/CSA and VHMW/CSA, as well as LMW/CSA and VHMW/CSA, showed distinct immunoprecipitin bands, respectively. The immunoelectrophoretic mobility of VHMW/CSA was similar to an alpha-globulin, whereas HMW/CSA and LMW/CSA migrated to the prealbumin region. CSA appeared in immunofluorescence of GW-39 tumor cells and in the goblet cells of human colon predominantly as a cell-surface component. Staining with periodic acid-Schiff and solubility characteristics of the CSA's suggest that they are glycoprotein in nature. These studies thus support the view that organ-specific and organ-associated antigens of the colon can be maintained and expressed in human colonic carcinomas, including the xenografted GW-39 human colonic tumor system.


Fetal antigens in human leukemia.

Immunization of BALB/c male mice with human peripheral leukemic blasts effectively reduced the later formation of syngeneic fetal liver, but not bone marrow hematopoietic colonies in the spleen when these mice were lethally irradiated and challenged i.v. Fetal antigen was detected in 6 of 6 lymphocytic leukemic patients and in 4 or 8 myelocytic leukemia patients and was correlated with low cellular levels of sialic acid. A rabbit antiserum ot BALB/c 15-day fetal liver cells labeled only 0 to 2% of normal donor peripheral leukocytes in indirect immunofluorescence but reacted with 10 to 21% of leukemic peripheral blasts. Active disease bone marrow on the same patients gave 7 to 40% fluorescent cells. Two remission bone marrow smaples were negative and 1 had 44% fluorescent cells. Using this antiserum coupled to sepharose, affinity column separation of KCl extracts from mouse and human fetal liver and from chronic lymphocytic leukemia has produced 4 common protein bands (identifiable on polyacrylamide gel electrophoresis).


Human chorionic gonadotropin in human neoplastic cells.

Peroxidase-labeled antibody against the beta chain of human chorionic gonadotropin was used to demonstrate that 25 human malignant tumors contained this antigen. The antigen was localized both in the cytoplasm and on the surface of the malignant cells. Human chorionic gonadotropin may be responsible for both selective maternal immuno-suppression by fetal tissue and host immunosuppression by tumors.


Physical characterization and structural studies of the carcinoembryonic antigen.

Knowledge of the structure of carcinoembryonic antigen (CEA) is essential if we are to understand the relationship of this molecule to similar, possibly cross-reacting, molecules present in nonmalignant states. Electron microscopy shows that at neutral pH, CEA particles consist of homogeneous, morphologically distinctive, twisted rod-shaped particles, about 9 X 40 nm. The carbohydrate structure of CEA has been studied by periodate oxidation. All the N-acetylneuraminic acid and fucose and a portion of the galactose and mannose were destroyed by the first periodate treatment without altering immunological activity. N-Acetylneuraminic acid was shown to be linked to galactose since its prior removal with neuraminidase led to an equivalent increased destruction of galactose by one treatment with periodate. Significantly, even after 100% of the fucose and N-acetylneuraminic acid, 75% of the galactose, and 50% of the N-acetylglucosamine and mannose were destroyed by serial periodate oxidation (Smith degradation), the remaining portion of the CEA molecule lost no more antigenic activity than did control samples where periodate was omitted. No carbohydrate was lost or destroyed in the control reaction.


Immunochemical studies on carcinoembryonic antigen-reactive glycoproteins from carcinomas of the colon and breast separated by concanavalin A affinity chromatography.

Analysis of carcinoembryonic antigen (CE)-reactive glycoproteins from liver metastasis of primary colon and breast tumors and from primary breast tumors has been carried out by affinity chromatography on concanavalin A (Con A)-Sepharose. Three CEA-reactive glycoproteins from colon tumors (liver metastasis) with different binding capacity to Con A have been separated and further purified by gel filtration. Of the 3 CEA-reactive glycoproteins, 1 of them did not bind to Con A. Both Con A-binding and nonbinding CEA-reactive glycoproteins were immunologically indistinguishable when tested with a reference goat anti-CEA (ACE, 67-70; Dr. C.W. Todd and Dr. M.L. Egan), as well as with a variety of rabbit anti-CEA and anti-CEA (nonbinding) prepared in this laboratory. Carbohydrate analysis showed that mannose content of different purified CEA preparations or nonbinding CEA did not differ appreciably. N-Acetylglucosamine content of purified CEA preparations, however, varied considerably, suggesting that this sugar may impart the specificity of binding of CEA to Con A. The purified CEA preparations differed in their ability to inhibit the binding of 125l-labeled CEA to goat anti-CEA. One of the purified CEA preparations had 3- to 8-fold greater inhibitory capacity when compared to other preparations and shared a partial identity with a glycoprotein present in the extracts of fetal colon. The glycoprotein extracts of primary breast tumors did not contain a CEA that was immunologically identical to CEA present in colon tumors, whereas the liver metastasis of primary breast tumors showed several CEA-reactive glycoproteins as judged by radioimmunoassay. However, these CEA-reactive glycoproteins did not have any antigenic relationship with CEA from colon tumors when tested by double diffusion and immunoelectrophoresis. In conclusion, when Con A affinity chromatography of tumor glycoproteins is carried out under defined conditions and with the use of appropriate antisera, it is possible to delineate the presence or absence of CEA in tumors of nonentodermal origin.


Biochemical changes in premalignant intestines.

The immunological properties of thymidine kinase from a variety of human tumors suggest that the form of the tumor enzyme resembles that found in the placenta and in the nondividing colonic flat mucosa. To examine the placenta-like characteristics of tumor thymidine kinase, the jejunum and colon from rats ranging in age from fetal to old and from animals treated with dimethylhydrazine (DMH), an intestinal carcinogen, have been studied. In normal jejunum, thymidine kinase activity decreased rapidly with age. Both the activity and the response to phospholipase C and to mercaptans in DMH-induced tumors resembled that of fetal gut, while those in abnormal appearing DMH-treated jejunum were intermediate between normal control of the same age and tumor. Similar but less pronounced changes were seen in the colon. In the jejunum, the level of another enzyme normally associated with rapid cell division, ornithine decarboxylase, was found to be over 100 times that of the liver, colon, and stomach. Treatment of the animals with acetylaminofluorene and with DMH resulted in elevated levels of the enzyme in liver and in colon, respectively, but had little effect on this enzyme in other tissues. The data presented indicate that there were premalignant changes in the levels of both of these enzymes in target tissues of animals treated with carcinogens.


Expression of early fetal antigens on transformed mouse cells.

We have used immunoprecipitation of radioactivity from labeled cell membrane preparations to study further the association between the histocompatibility-2 (H-2) peptides and a tumor-associated embryonic antigen (Ag I) on the surface of L-cells. Since earlier studies had shown that Ag I and H-2 co-cap on L-cells, the present study was initiated to test the hypothesis that Ag I presents an altered H-2 peptide expressed on transformed cells. Indeed, sodium dodecyl sulfate-gel electrophoresis of Ag I shows two major peaks at approximately 110,000 and 40,000 daltons, the latter co-migrating with the major H-2 peptide. However, further analysis using sequential immunoprecipitation indicates that the molecules reacting with antisera to H-2 and to Ag I are separate entities, precipitating independently of one another. This has led us to postulate that Ag I and H-2 participate in a unique kind of association in the cell membrane, that is, that the bonds between them which allow them to co-cap may be in the lipid bilayer itself, and that these bonds are distrupted upon solubilization of the membrane.


The isolation and characterization of tumor-specific antigens of rodent and human tumors.

Putative tumor-specific transplantation antigens (TSTA) from both a carcinogen-induced rodent tumor (MC-1) and 2 human tumors were purified. The antigens were solubilized from the tumor cell membranes by limited papain digestion in a manner similar to that described for the isolation of normal histocompatibility antigens. The antitumor immune response of the tumor-bearing host was used to monitor the purification of the putative TSTA in both the rodent and human tumor systems. In the case of the rodent tumor, a major step in the purification of the TSTA involved affinity chromatography on Sepharose beads coupled to autologous antitumor antiserum. A comparable procedure was utilized in the purification of the TSTA from human tumors by using affinity chromatography on anti-human beta2-microglobulin antiserum coupled to a solid phase. The data obtained indicate that the TSTA of human tumors contains a beta2-microglobulin chain that is immunochemically identical with, and very similar in size to, that found in normal human histocompatibility antigens. A subunit of similar size was also identified in the carcinogen-induced rodent tumor. These results suggest that the TSTA in both humans and rodents may well be altered histocompatibility antigens.


Cell surface alterations on colon adenocarcinoma cells.

By the use of five independent techniques, cell surface alterations distinctive of malignant as compared to normal colon cells were detected on in vivo surgical specimens and on cultured cell lines established in our laboratory. The findings, which were distinctive of the malignant as compared to the normal cell included: (a) polymorphism of surface microvilli on scan electron microscopy; (b) decreased susceptibility to infection with vaccinia and reovirus, but not to herpes, adeno- or echovirus: (c) production of large quantities of carcinoembryonic antigen; (d) presence of specific membrane proteins on sodium dodecyl sulfate-polyacrylamide gel analysis of plasma membranes purified from cell homogenates by ultracentrifugation in polyethylene glycol-dextran partitions; and (e) reaction with specific, cytotoxic, rabbit heteroantisera. Solubilized extracts of the malignant cells formed precipitin lines with the heteroantisera, suggesting that the distinctive antigens could be released from the cell surface. These results suggest that human colon carcinomas bear tumor-distinctive proteins and offer the prospect of specific immunodiagnostic reagents and immunotherapeutic tools.


Angiogenesis induction by tumors, embryonic tissues, and lymphocytes.

Although it is well established that tumors can induce endothelial cell mitosis and angiogenesis leading to extensive neovascularization, little is known concerning the spectrum of normal cells or tissues that may have similar inductive capabilities. A variety of developmentally active tissues were tested: mouse embryonic organ rudiments, mouse and rabbit trophoblast and placenta, and blastemal fragments obtained from regnerating deer antlers. Assays for angiogenesis included grafts made to the chorioallantoic membrane of chick embryos and grafts placed intracorneally in adult rabbit eyes. Virtually all of these test tissues were negative, the only  exceptions being some antler grafts and some grafts obtained from metanephric tubules of the embryonic mouse kidney.


Cell cycle dependency of tumor antigens.

There is now substantial evidence that suggests that tumor antigen expression is cell cycle dependent. This information has been obtained from a wide variety of tumor systems. The G1 phase of the cell cycle has been implicated by a number of investigators as the point of maximal antigen appearance. In this presentation, we are concerned with a human sarcoma-associated tumor antigen and its cell cycle-dependent appearance. Sarcoma-associated tumor antigen is a membrane antigen present on cultured human neurosarcoma cells (T2-cells). Although the underlying mechanisms responsible for cell cycle-dependent expression are unclear, several possible interpretations are offered.


[Condensation of D-glycosamines with proteins].

4-O-beta-D-Galactopyranosyl-alpha,beta-D-glucopyranosylamine (lactosylamine), beta-D-gluco-, alpha- and beta-D-galacto-, and beta-D-manno-pyranosylamines were bound to the carbodiimide-activated carboxyl groups of lysozyme. Of the 11 free carboxyl groups of the protein, approximately 3 were substituted by alpha,beta-lactosylamine, and approximately 2 by the monohexosylamines. One of the 4 glycopeptides isolated from the tryptic digest of the lysozyme-lactosylamine conjugate was identical to synthetic 1-N-L-leucinoyl-4-O-beta-D-galactopyranosyl-beta-D-glucopyranosylamine, indicating the substitution of the carboxyl group of the C-terminal leucine residue. The isolation of a glycopeptide containing the aspartic acid residue in position 117 indicates that the second alpha,beta-lactosylamine residue is linked to the carboxyl group of this amino acid. Both of the 2 other glycopeptides contain the same free carboxyl groups (one glutamic and two aspartic acid residues in positions 35, 48, and 52, respectively). The third alpha,beta-lactosylamine residue seems to be linked to one of these carboxyl groups.


[Lengthening of the peptide chain at the C-terminal end of a glycosylamino acid].

An O-glycodipeptide was synthesized by lengthening the peptide chain on the C-terminal side of a glycosylamino acid unit. N-(Benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-L-threonine o-nitrophenyl ester and pentachlorophenyl ester were condensed with glycine benzyl ester to give both the same glycodipeptide, [N-(benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-L-threonyl]glycine benzyl ester (9). The two anomers, N-(benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-alpha- and beta-D-glucopyranosyl)-L-threonine pentachlorophenyl ester were also prepared. 1H-N.m.r. studies ascertained the structure and anomeric configuration expected.


Characterization of the fragments obtained by enzymic and alkaline degradation of rice-bran proteoglycans.

Repeated digestion of the proteoglycan B from rice bran with Pronase and hemicellulase yielded two types of glycopeptides. The first group, obtained from the third enzymic digest, was a type of low molecular-weight glycopeptide and was fractionated into two subfractions by paper electrophoresis. The second group, obtained from the fourth enzymic digest, was a mixture of glycopeptides of relatively high molecular-weight and was fractionated into three subfractions by gel filtration on Sephadex G-75. Alkaline degradation of these glycopeptides or the intact proteoglycan A yielded a sugar-amino acid compound, whose structure was established as O-alpha-L-arabinofuranosyl-hydroxyproline. These results indicate that the carbohydrate-protein linkage of the rice-bran proteoglycan is an O-glycosyl linkage between L-arabinose and hydroxyproline.


The limit dextrinase from malted sorghum (Sorghum vulgare).

A limit dextrinase, free from contaminating carbohydrases, has been purified from malted sorghum flour. The enzyme readily hydrolysed alpha-limit dextrins having maltosyl or maltotriosyl side-chains, pullulan, and amylopectin beta-limit dextrin. Glycogen beta-limit dextrin and amylopectin were more slowly hydrolysed, the detection of the hydrolysis of amylopectin being dependent on enzyme concentration. No significant debranching of glycogen could be detected.


Autonomic neural control of cardiac rhythm: the role of autonomic imbalance in the genesis of cardiac dysrhythmia.

Cardiac dysrhythmias result from abnormalities in rate, regularity, or sequence of cardiac activation, and because of direct actions of the autonomic nervous system upon each of these properties, imbalance in this system may play an important role in the genesis of cardiac dysrhythmia. A canine model has been developed in which the extrinsic innervation of the heart is ablated with the exception of the ventrolateral cardiac nerve. This nerve is distributed primarily to the inferior atrial, AV junctional, and ventricular tissues. Following recovery from surgery, the animal is placed on a treadmill and required to perform strenuous exercise. In all of six animals which sustained repeated exercise testing over periods of 4-12 months, dysrhythmias of varying complexities were elicited. None appeared in parallel experiments conducted in control or sham-operated animals. The dysrhythmias consisted of supraventricular, AV junctional, or ventricular tachycardias with occasional premature atrial or ventricular systoles. The dysrhythmias were not influenced by atropine but were generally controlled by propranolol.


Role of the nervous system in experimentally induced arrhythmias.

The purpose of our studies was to examine the role of the nervous system in arrhythmias produced by digitalis overdose and coronary artery occlusion in the cat. This was done by observing the effect of these arrhythmogenic procedures on cardiac efferent neural activity and then determining whether any observed alteration in neural activity contributed to the cardiac rhythm disturbances evoked by digitalis and coronary artery occlusion. Our data indicate that both procedures used to evoke arrhythmias activate each division of the autonomic nervous system. Activation of the sympathetic nervous system resulted in a deleterious effect on cardiac rhythm whereas activation of the parasympathetic nervous system, in general, resulted in a beneficial effect on cardiac rhythm. With coronary occlusion, the role exerted by the nervous system depended on the anatomic location of the involved myocardium. Studies directed at elucidating the mechanisms whereby the nervous system caused cardiac rhythm disturbances indicated that there may be an important difference between the antiarrhythmic efficacy of beta-adrenergic blockade and bilateral stellate ganglionectomy. The latter procedure proved to be a more effective way of removing deleterious sympathetic neural effects on the heart. In conclusion, our findings suggest that the development of new drugs for treating arrhythmias resulting from digitalis and coronary occlusion should be aimed at finding drugs that act to either depress central sympathetic outflow or enhance parasympathetic effects on the ventricle.


Neural basis for the genesis and control of digitalis arrhythmias.

The pharmacokinetics of ouabain associated with toxicity were studied in the cat and the guinea pig both in vivo and in vitro using ouabain-H3. After spinal cord transection a higher dose of ouabain was required to reach the lethal endpoint. This intervention also increased the myocardial and serum levels associated with toxicity were studied in the cat and the guinea pig both in vivo and in vitro using ouabain-H3. After spinal cord transection a higher dose of ouabain was required to reach the lethal endpoint. This intervention also increased the myocardial and serum levels associated with death. These findings were corroborated in experiments using digitoxin H3. In vitro, substantially higher ouabain tissue contents were associated with a lethal event. In addition, in cats and guinea pigs, the lethal myocardial ouabain content did not change when the infusion rate of ouabain was varied in vivo or the perfusate ouabain concentration was changed in vitro. In vivo, propranolol increases the myocardial ouabain content associated with death to in vitro levels. In vitro, the drugs prolongs the time to death by retarding the myocardial uptake of ouabain. These data suggest that the toxic effects of ouabain in the whole animal are largely neural and in the isolated heart, substantially myocardial.


Central nervous system control of cardiac rhythm.

Stimulation of sites in the midbrain reticular formation and in the posterior hypothalamus of the cat resulted in a large to modest rise of arterial pressure and the induction of cardiac dysrhythmias. Most frequently, these arrhythmias developed upon cessation of brain stem stimulation but also occurred during the stimulus period in 5 of 23 cats studied. The arrhythmias disappeared upon cooling and reappeared upon rewarming the vagus nerves. The ventricular dysrhythmias also were abolished by methylscopolamine, by bilateral vagatomy, or by extirpation of the stellate ganglia. Simultaneous stimulation of both distal end of the cut right vagus nerve and the decentralized right stellate ganglion caused arrhythmias similar to those observed after diencephalic stimulation. These data are interpreted to indicate that the cardiac arrhythmias evoked by brain stem stimulation result from the interplay of both sympathetic and parasympathetic influences on the heart. The response patterns of a population of medullary neurons activated by carotid sinus nerve stimulation were modified by condition stimuli to posterior hypothalamic sites. From studies on unit activity of brain stem areas known to participate in cardiovascular adjustment, a schema is proposed of hypothalamic-medulla interaction as a central mechanism that may account for the development of ventricular arrhythmias.


Persistence of a basal lamina-like structure following DOCA-induced myofibrillar degeneration in rats.

It is known that the myocardial necrosis of potassium depletion heals by reconstitution and not by scarring, when animals are replenished with potassium. In the course of studies on DOCA-induced myofibrillar degeneration, we found that a membrane-like structure persisted in the area where the disintegrating myofiber was being removed by macrophages. This structure resembled the basal lamina, and enclosed a space in which macrophages with phagosomes containing disintegrating myofibral constituents were seen in association with undifferentiated cells. We postulate that this basal lamina-like structure along with the undifferentiated cells, play a role in the reconstitution of the myocardium during the stage of potassium repletion, and that the scaffolding by basal lamina may be effective in myocardial reconstitution.


Familial cardiomyopathy.

Over three generations, several members of a family suffered from cardiomyopathy exhibiting, primarily, rhythm and conduction disturbances and, eventually, congestive heart failure. Certain serum enzymes (SGOT, SGPT, LDH) were elevated. The clinical examination indicated a progressive deterioration with increasing age of the patients, a more serious course in the male members of the family, and the frequent association of mental retardation.