extfieldsofstudy: Medicine Biology

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Misfolded Proteins are Competent to Mediate a Subset of the Responses to Heat Shock in Saccharomyces cerevisiae 1

To this possibility, we the effect of misfolding on gene in the absence of temperature changes. The imino acid analogue azetidine 2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We find that adding AZC to yeast, at sublethal concentrations sufficient to arrest proliferation, selectively induces expression of heat shock factor-regulated genes to a maximum of 27 fold and that these inductions are dependent on heat shock factor. AZC treatment also selectively represses expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20 fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins: induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activates heat shock factor, but treatment with canavanine, an arginine analogue less potent than AZC at misfolding proteins, does not. However, misfolded proteins do not strongly induce the STRE regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.

Eukaryotic cells respond to heat shock by the induction of a conserved set of proteins, the heat shock proteins, via transcriptional activation of the corresponding genes (1). In the budding yeast, Saccharomyces cerevisiae, two distinct promoter elements mediate transcriptional activation in response to heat shock (reviewed in 2). Heat shock elements (HSEs) are found upstream of many heat-induced genes, e.g., HSP42 and SSA4. Heat-shock factor binds to HSEs and is required for the induction of HSE-driven genes in response to heat shock. Stress response elements (STREs) are also found upstream of many heatinducible genes, e.g., CTT1 and DDR1, and bind the transcription factors Msn2 and Msn4 (2). Loss of both transcription factors compromises heat shock-induced expression of STREcontaining genes. Some heat shock-inducible genes contain both HSEs and STREs in their promoters, e.g., HSP12, HSP30 and HSP104. The HSE and STRE regulons constitute the majority, if not all, of the genes that are specifically induced by heat shock (3).
Rapid upshifts in temperature within the permissive growth range of yeast (so-called "temperature upshifts"), e.g., 23 to 36 degrees Celsius, result in the transient and selective induction of the heat shock genes (both HSE-and STRE-containing) and in the transient and selective repression of the 137 ribosomal protein genes (4 and references therein). Heat shocks to non-permissive temperature (e.g., to 42 degrees Celsius) also cause a global repression of gene expression not seen upon temperature upshift (4). Repression of the ribosomal protein genes by heat shock is dependent on activation of heat shock factor,.
Because these genes do not contain HSEs, the repression is thought to be indirect (4).
The current model for how cells sense heat shock is as follows (6, and references therein). Heat shock is proposed to cause the thermal misfolding of a fraction of cell protein.
Because activation of heat shock factor requires protein synthesis, it is thought that nascent proteins are the most susceptible to thermal denaturation. Misfolded proteins then bind to cytoplasmic Hsp70 chaperones. Prior to heat shock, these chaperones are believed to equilibrate between being bound to heat shock factor (and inactivating it) and being free in the cytoplasm. Because misfolded proteins bind Hsp70s very tightly, their accumulation upon heat shock is proposed to titrate Hsp70 chaperones resulting in liberated and active heat shock factor. Consistent with this model, misfolded proteins have been detected in by guest on March 23, 2020 http://www.jbc.org/ Downloaded from

Materials and chemicals
All chemicals were from Sigma-Aldrich Company (Dorset, UK) Components of growth media were from Becton Dickinson (Sparks, Maryland, USA) and Fisher Scientific UK (Leicester, UK). D/L-AZC was used throughout this work but L-AZC is the active agent in this racemic mixture 3 . Any given concentration of AZC herein refers to the concentration of the racemic mixture.

Plasmids, yeast strains and manipulations
Liquid media, both rich and minimal, were prepared as described previously (16).
Solid media contained 2% agar. Strains were routinely grown on YPD agar plates at 25 o C.
Liquid cultures were grown in YPD broth.

Northern analysis
Extraction of total RNA was performed as previously described (Ogas et al, 1991

RESULTS
In this study, we set out to determine if AZC treatment mimics the effect of heat shock on genome-wide gene expression and, if so, to explore the mechanism by which the analogue causes these effects.
Laboratory strain backgrounds differ in their sensitivity to AZC, as judged by colony formation on plates (15) 2 . The concentrations used herein are those just sufficient to permanently inhibit proliferation of each strain background under study, unless stated otherwise. All experiments were conducted in the presence of normal amounts of proline in the growth medium.
We determined the expression changes of the 6129 protein-encoding genes by microarray analysis (see experimental procedures) using wild-type W303-1A cells treated with a growth-inhibitory concentration of AZC (50 mM) for five hours. We have also performed an equivalent analysis after one hour of AZC treatment (50 mM). The result of the latter experiment was qualitatively similar to the five hour data set but with less potent expression changes 2 . Our subsequent analysis has therefore focused on the five hour data set.

AZC treatment does not cause starvation.
AZC is an analogue of proline and may thereby interfere with proline uptake or metabolism, resulting in starvation. Nutrient-starved cells or cells treated with rapamycin, an inhibitor of the TOR proteins, stop proliferation in the G1 phase of the cell cycle and enter quiescence-like, non-proliferating states (20) superficially reminiscent of AZC-arrested cells (15). We find by microarray analysis that AZC treatment ( We determined the correlations between the microarray-derived expression profiles of AZC-arrested cells (5 hr, this work) and temperature-upshifted cells (23 o C to 37 o C for 10 min, 20 min and 40 min) (18). This analysis focused on those genes (2470 in total) of known function because data for this subset upon temperature upshift are publicly available (18).
We find that the correlation between AZC (5 hr) and temperature upshift microarray data sets is highest for the 20 min timepoint of temperature upshift, peaking at 0.576. This correlation compares well with those between temperature upshift timepoints: 10 min versus 20 min -correlation of 0.793; 10 min versus 40 min -correlation of 0.582 (18) 3 . These data suggest that AZC treatment selectively causes the majority, but not all, of the gene expression changes characteristic of temperature upshift. We conclude that AZC treatment partially mimics temperature upshift.
AZC treatment selectively causes heat shock factor-dependent gene expression changes.
What genes are selectively induced by AZC treatment? Expression of a subset of genes was strongly induced by treatment with AZC (see Table 1  Activation of heat shock factor directly or indirectly causes repression of the ribosomal protein genes (4). If AZC treatment indeed activates heat shock factor, then we expect treatment with the analogue to also repress expression of the ribosomal protein genes.
Expression of a subset of genes (293 in total) was repressed by a factor of three or more (to a maximum of 20 fold) by AZC treatment (see Table 1 for a partial list and supplementary data for the complete list). Of the 50 genes of known function that are repressed most strongly by AZC treatment, 47 are also strongly repressed by temperature upshift (18). The majority of these (42 out of 47) encode ribosomal proteins and repression of these genes by severe heat shock is dependent on heat shock factor (4). Our data suggest that AZC treatment, like temperature upshift, selectively activates heat shock factor and thereby causes increased expression of HSE-containing genes and repression of the ribosomal protein genes.
Of the 50 genes of known function whose expression is repressed most strongly by temperature upshift (18), 46 are also strongly repressed by AZC treatment. Again, the majority of these genes encode ribosomal proteins. Thus AZC treatment selectively causes the vast majority of the repressions caused by temperature upshift.

AZC treatment fails to strongly induce expression of the STRE regulon.
Almost all of the genes whose expression is induced by temperature upshift are (3). We conclude that the STRE regulon is at best weakly activated by AZC treatment. This possibility is supported by our previous finding that AZC treatment fails to cause accumulation of glycogen and trehalose, phenomena dependent on activation of the STRE pathway (15). We conclude that the STRE regulon is much less sensitive to AZC treatment than is the HSE regulon.
To a first approximation, temperature upshift selectively induces the STRE regulon and the HSE regulon (and thereby represses expression of the ribosomal protein genes), whereas AZC treatment selectively induces only the latter.

AZC treatment causes few gene expression changes not caused by temperature upshift.
Although incorporation of AZC into any given protein molecule is likely to cause dysfunction of that protein, the efficiency of incorporation of AZC in place of proline in our experiments is likely to be low (15) (see discussion). Hence, most molecules of any particular protein in AZC-treated cells are unlikely to contain AZC and thus are functional.
However, proteins that are large, proline-rich and short-lived are most likely to incorporate at least one residue of AZC in place of a proline. Thus AZC treatment may preferentially inactivate a particular subset of proteins within the cell and thereby directly cause gene expression changes not caused by temperature upshift. Surprisingly, we find only a handful of genes in this set: CUP1-1, CUP1-2 and ICS3 are induced by AZC treatment and not temperature upshift; ACS2, RPN8, CTS1 and BUD2 are repressed by AZC treatment but not by temperature upshift (18, see Table 1 for a partial list and supplementary data for the complete list of genes whose expression is affected by a factor of 3 or more by AZC treatment). Therefore, the majority of the gene expression changes caused by AZC treatment are not due to selective inactivation of any particular subset of proteins by the analogue. If AZC treatment and temperature upshift cause widespread misfolding of cell proteins, then we would expect these treatments to activate the unfolded protein response pathway of the endoplasmic reticulum (the ER-UPR pathway). This signalling pathway is activated by the accumulation of misfolded proteins in the endoplasmic reticulum and promotes transcription (and thus expression) of genes containing unfolded protein response elements (UPREs) in their promoters (19). Significantly, we find that genes that are strongly induced by the ER-UPR pathway, e.g., EUG1, PDI1, and LHS1, are not strongly induced by AZC treatment (1.15-fold, 1.7-fold and 1.63-fold respectively) 2 . Expression of these genes is also not strongly induced by temperature upshift (18). These findings are consistent with AZC treatment and temperature upshift causing only a low level of protein misfolding in the endoplasmic reticulum (and presumably throughout the cell).

AZC treatment strongly and robustly causes heat shock factor-dependent gene expression changes.
We wished to confirm the salient features of the gene expression changes detected from microarray profiling by Northern blot analysis. We also wished to compare the magnitudes of these expression changes with those caused by temperature upshift. For this analysis, we used a wild-type haploid strain of the S288C background whose proliferation in YPD is inhibited by 10 mM AZC (15). We determined the expression level of the following genes as a function of time after addition of AZC (10 mM) or upon temperature upshift However, the promoters of these genes fall into two classes, those containing potential binding sites for the Rap1 transcription factor and those containing potential binding sites for the Abf1 transcription factor. We included representatives of both subclasses for completeness. Equal amounts of total RNA were loaded in each lane, and blots were hybridised with the same probe at the same time, and exposed to the same film for equal amounts of time for each probing.
The results of the Northern analysis are shown in Fig. 1a Figure 2a,b, we find that these HSE-containing genes are indeed transiently induced in this strain background in response to AZC treatment.
Furthermore, we find that expression of HSP42 is induced more strongly in the EXA3-1 mutant compared to the wild-type strain in response to both AZC treatment and temperature upshift. Expression of HSP42 is also more persistent in the mutant in response to both treatments. Although there may be subtle effects of the EXA3-1 mutation on the extent of SSA4 induction in response to both AZC treatment and temperature upshift, expression of this gene is clearly more persistent in the mutant in response to both treatments. The EXA3-1 mutation in HSF1 thus affects the extent or persistence, or both, of induction of HSP42 and SSA4 in response to AZC treatment and temperature upshift. These data indicate that AZC treatment and temperature upshift cause induction of HSP42 and SSA4, and by inference all HSE-containing genes, by the same mechanism, namely activation of heat shock factor.
The induction of HSP42 and SSA4 by AZC treatment is clearly transient for the wildtype strain used in Figure 2, in contrast to the equivalent data for the S288c wild-type strain shown in Figure 1. The persistence of heat shock factor activation in response to AZC treatment in these strain backgrounds clearly parallels the persistence of proliferation arrest caused by the analogue, which gratifyingly is also dependent on heat shock factor (15).
Although the reason for the different responses of these two strain backgrounds to the analogue are not known, it is likely to be due to some combination of differences in the rate of uptake or efflux of the compound, to different efficiencies of incorporation of the analogue into protein or to different capacities to degrade analogue-containing peptides.
From our data in Figures 1 and 2, we cannot compare the relative expression levels of HSP42 and SSA4 between these two strain backgrounds since different batches of labelled probes were used for each experiment.

Induction of HSP12 by AZC treatment is partly dependent on Msn2 and Msn4.
From the microarray analysis above, STRE-containing genes are at best poorly induced by AZC treatment (  Figure 1a,b), we set out to determine if Msn2 and Msn4 contribute to induction of HSP12 upon AZC treatment. We find that the induction of HSP12 by AZC treatment and by temperature upshift is significantly reduced in a strain deleted for both MSN2 and MSN4 relative to its congenic wild-type strain (Figure 3a,b). We conclude that Msn2 and Msn4 (and by inference the STRE regulon) may indeed be activated by AZC treatment, but only partially.

Induction of HSP42 by AZC treatment is dependent on protein synthesis.
If AZC exerts its effects in vivo by misfolding proteins into which it is incorporated, then the expression changes caused by AZC treatment should require ongoing protein synthesis. We therefore determined the effect of cycloheximide addition on the ability of AZC treatment to induce expression of HSP42. Treatment of S288c wild-type cells with cycloheximide alone or vehicle alone does not alter expression of HSP42. In contrast, we find that the presence of cycloheximide prevents induction of HSP42 by AZC treatment (Figure 4a). We infer that ongoing protein synthesis is required for AZC treatment to induce the HSE regulon, consistent with the analogue functioning via misfolding nascent proteins into which it is incorporated.

Ethanol treatment mimics AZC treatment.
If AZC acts via misfolding cellular proteins, then we expect other treatments that misfold proteins in the cell to induce the same spectrum of gene expression changes as that caused by AZC treatment.
Ethanol can disrupt protein folding by a mechanism distinct from that of AZC (5).
The results of the Northern analysis of ethanol-treated cells are shown in Fig 4a. These blots, and those for canavanine-treated cells (see next section, Figure 4b) were prepared identically to, and probed with the same batch of labelled probe at the same time and exposed to the same film for the same length of time as, the blots shown in Figure 1a,b. Thus, the data shown in Figures 1 and 4 are directly comparable. The concentration of ethanol used in this experiment (8% v/v) is just sufficient to stop proliferation of the strain used (S288C wild type) 2 . We find that ethanol treatment mimics AZC treatment: 1) in strongly inducing the expression of genes regulated by heat shock factor, e.g., HSP42, SSA4, HSP12 and HSP30; 2) in failing to strongly induce CTT1 (a STRE-driven gene) in agreement with the very weak activation of STRE-driven genes by ethanol reported previously (26); 3) in failing to repress ACT1 (i.e., no global repression); 4) in strongly repressing the expression of the ribosomal protein genes tested. We conclude that ethanol and AZC treatments, both of which can misfold proteins but by very distinct mechanisms, cause similar gene expression changes attributable to activation of heat shock factor.

Canavanine treatment does not mimic AZC treatment.
Canavanine is an arginine analogue that, like AZC, is incorporated into protein competitively with the corresponding natural amino acid (27). Canavanine differs from arginine in the structure of its sidechain and, as such, is not expected to significantly alter the conformation of the polypeptide backbone into which it is incorporated (relative to the same protein containing arginine at the equivalent position(s)). Thus, treatment with canavanine is not expected to cause protein misfolding, at least not to the same extent as does AZC treatment.
We examined the effect of canavanine at a sub-lethal concentration (10 mM) just sufficient to inhibit cell proliferation, on gene expression by Northern analysis. Our results are shown in Figure 4b. In contrast to ethanol and AZC treatments, canavanine treatment does not significantly induce expression of the HSE-containing genes HSP42 and SSA3.
Canavanine treatment also fails to significantly repress expression of the ribosomal protein genes. We infer that canavanine treatment does not strongly activate heat shock factor.
Canavanine treatment also fails to induce expression of CTT1 and is thus unlikely to strongly activate the STRE regulon. It should be noted that in our experiments, both AZC and canavanine are present in the growth medium at concentrations sufficient to inhibit proliferation 2 , yet only AZC treatment induces the HSE regulon. Thus, activation of heat shock factor by AZC treatment is not a consequence of growth inhibition per se. Rather, the ability of the analogues, AZC and canavanine, to activate heat shock factor correlates with their relative capacity to misfold proteins into which they are incorporated.  (HSP12 and HSP30) but too weakly to drive expression of genes dependent on one or other system.

AZC causes protein misfolding in vivo.
Multiple lines of evidence indicate that AZC exerts its effects on yeast cells via Finally, ubc4.ubc5 mutants, which are defective in the ubiquitin-dependent degradation of short lived and analogue-containing polypeptides, are hypersensitive to AZC treatment (15).
The simplest explanation for this observation is that, at a given concentration of AZC, the amount of analogue-containing protein in the cell is higher when these proteins are stable than when these proteins are unstable. Thus, a concentration of AZC that is insufficient to arrest cell proliferation in a wild-type cell would be sufficient to arrest a cell lacking Ubc4 and Ubc5. Taken together, these arguments strongly indicate that AZC acts via incorporation into cellular protein.
Given that incorporation of AZC into protein is known to cause reduced thermal stability or misfolding (12)(13)(14), the effects of the analogue on gene expression are most likely due to its misfolding proteins. Two lines of evidence support this notion. First, induction of the HSE regulon is also strongly and selectively caused by treatment with ethanol, another agent capable of misfolding proteins but by a mechanism different to that of AZC. Second, canavanine, an arginine analogue that is incorporated into protein competitively with arginine, does not induce the HSE regulon, whereas AZC does so efficiently. Canavanine incorporation is not expected to disrupt protein folding as efficiently as does incorporation of AZC (27). Thus, the relative of the analogues to induce the HSE regulon correlates with their capacity to misfold proteins into which they are incorporated.

AZC treatment selectively activates heat shock factor.
AZC treatment selectively causes the gene expression changes attributable to activation of heat shock factor. First, the expression level of only a small fraction of the protein-encoding genes are altered by a factor of three or more after five hours of treatment with an inhibitory concentration of AZC (8.2% affected in total: 3.5% induced; 4.7% repressed). Hence, AZC treatment does not cause any global changes in gene expression, but selectively affects expression of a discrete subset of genes. Second, HSE-containing transcripts predominate amongst those induced by AZC treatment. Third, the ribosomal protein genes (and co-regulated genes encoding components of the translation apparatus) comprise the vast majority of the genes that are strongly repressed by AZC treatment.
Repression of the ribosomal protein genes by heat shock is known to be dependent on activation of heat shock factor (4).
We have confirmed that AZC treatment activates the HSE regulon (induction of HSE-containing genes and consequent repression of the ribosomal protein genes) by Northern analysis. Furthermore, we find that AZC treatment activates the HSE regulon as strongly if not more strongly than does temperature upshift. We have also shown that a mutation in heat shock factor affects induction of the HSE-containing genes HSP42 and SSA4 in response to AZC treatment. Critically, the mutation alters the induction of these genes in the same way in response to either AZC treatment or temperature upshift. Thus, by guest on March 23, 2020 http://www.jbc.org/ Downloaded from AZC treatment strongly and selectively induces the HSE regulon by the same mechanism as does temperature upshift, namely by activating heat shock factor.

Misfolded proteins are competent to be mediate selective activation of heat shock
factor in response to heat shock.
Based on the above arguments, we conclude that the misfolding of a fraction of cell protein in the absence of temperature change mimics heat shock in selectively and strongly activating heat shock factor. Therefore, misfolded proteins illicit the appropriate cellular response, and do so sufficiently strongly and selectively, for them to be intermediates in the cellular response to heat shock. Given that misfolded proteins are known accumulate in heat shocked cells (7), They are competent to mediate at least part of the cellular response to heat shock.
Unfortunately, it is not yet known if misfolded proteins are kinetically competent to be intermediates in the heat shock response, i.e., that misfolded proteins accumulate sufficiently rapidly upon heat shock and that misfolded proteins cause activation of heat shock factor sufficiently quickly. Although activation of heat shock factor is slow in response to AZC treatment, it is likely that equilibration of the analogue into the cellular proline pool prior to incorporation into protein is slow. The issue of kinetic competence remains unresolved.
If misfolded proteins are intermediates in the cellular response to heat shock, then heat shock factor must be very sensitive to protein misfolding in cytoplasmic space.
Temperature upshift is a very mild environmental change and is unlikely to cause extensive protein misfolding. In addition, AZC treatment at concentrations sufficient to activate heat shock factor does not appear to cause widespread protein dysfunction: 1) AZC treatment almost exclusively affects the expression of a small and discrete subset of genes that are also induced by temperature upshift; 2) AZC-arrested cells are viability (15); 3) AZC arrest is reversible (15); 4) AZC-arrested cells are responsive to subsequent treatments, e.g. heat shocks (15) and rapamycin 2 . Indeed, neither AZC treatment nor temperature upshift strongly activates the ER-UPR, even though both treatments should misfold proteins throughout the cell, including in the endoplasmic reticulum.
The fraction of protein containing AZC (when cells are treated with a concentration of the analogue just sufficient to activate heat shock factor) should constitute an upper limit for the fraction of cellular protein whose misfolding is just sufficient to activate heat shock factor. We are attempting to determine this number.
The STRE regulon is relatively insensitive to protein misfolding.
Although AZC treatment profoundly activates the HSE regulon and genes whose expression is dependent thereon (e.g., the ribosomal protein genes), it at best weakly induces the STRE regulon. This possibility is supported by our observation that AZC does not lead to the accumulation of glycogen and trehalose, a STRE regulon-dependent phenomenon (15), nor does it significantly activate expression of STRE-LacZ reporter constructs 3 .
However, AZC incorporation into cell protein does not appear to affect the activateability of the STRE regulon (15). Rather, AZC treatment simply fails to strongly activate this regulon.
The primary signal for activation of the STRE regulon by heat shock may be the misfolding of cellular protein, but with the STRE regulon requiring higher levels of protein misfolding than those sufficient to activate heat shock factor (and inhibit proliferation). Indeed, the timecourse of activation of CTT1 expression by temperature upshift parallels that of the heat shock factor-dependent transcripts, consistent with the notion of a common trigger.
Alternatively, the STRE pathway may primarily respond to heat-induced oxidative stress or some other stress that coincides with protein misfolding upon heat shock (2,5). It is clear that Msn2 and Msn4 contribute to the induction of HSP12 by AZC treatment. Given that the STRE regulon is activated by multiple stresses to the cell, it is possible that any partial activation of the regulon in response to analogue treatment is caused by an indirect mechanism, e.g., because of proliferation arrest. The mechanism by which the STRE regulon is activated by heat shocks remains elusive.

How do cells sense heat shocks?
Thermally-misfolded protein likely triggers activation of heat shock factor in response to heat shocks. The sensor for activation of the STRE regulon upon heat shock remains unclear. However, misfolded protein is not the sole sensor of heat shock in yeast.
The cell integrity pathway, which is required for acquired thermotolerance and for   Samples were prepared and analysed as for the AZC treatment above. Blots were probed with the same probes and exposed to the same film and for the same times for each probe.     Supplementary table legend   Table S1: Genes whose expression is altered upon AZC treatment by a factor of three or more. Microarray analysis was performed on wild-type cells (W303) grown to midlogarithmic phase in YPD at 30 o C and treated with AZC (50 mM) for 5 hours. Expression changes were determined by probing DNA microarrays as outlined in the main text.
Genes whose expression was induced or repressed by a factor of three or more (


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Aspergillus nidulans Catalase-Peroxidase Gene (cpeA) Is Transcriptionally Induced during Sexual Development through the Transcription Factor StuA

ABSTRACT Catalases, peroxidases, and catalase-peroxidases are important enzymes to cope with reactive oxygen species in pro- and eukaryotic cells. In the filamentous fungus Aspergillus nidulans three monofunctional catalases have been described, and a fourth catalase activity was observed in native polyacrylamide gels. The latter activity is probably due to the bifunctional enzyme catalase-peroxidase, which we characterized here. The gene, named cpeA, encodes an 81-kDa polypeptide with a conserved motif for heme coordination. The enzyme comprises of two similar domains, suggesting gene duplication and fusion during evolution. The first 439 amino acids share 22% identical residues with the C terminus. Homologous proteins are found in several prokaryotes, such as Escherichia coli and Mycobacterium tuberculosis (both with 61% identity). In fungi the enzyme has been noted in Penicillium simplicissimum, Septoria tritici, and Neurospora crassa (69% identical amino acids) but is absent from Saccharomyces cerevisiae. Expression analysis in A. nidulans revealed that the gene is transcriptionally induced upon carbon starvation and during sexual development, but starvation is not sufficient to reach high levels of the transcript during development. Besides transcriptional activation, we present evidence for posttranscriptional regulation. A green fluorescent protein fusion protein localized to the cytoplasm of Hülle cells. The Hülle cell-specific expression was dependent on the developmental regulator StuA, suggesting an activating function of this helix-loop-helix transcription factor.

Oxidative stress and the occurrence of reactive oxygen species is common to aerobically living organisms and might be deleterious for living cells (10,18). Reactive oxygen species are generated during normal cell metabolism and comprise superoxide, hydroxyl radicals, hydrogen peroxide, and singlet oxygen. All aerobically living organisms employ one or several systems to cope with these toxic substances. Catalases and peroxidases are most commonly used to transform the harmful oxygen compound H 2 O 2 into harmless products. Catalases are heme-containing enzymes, which convert H 2 O 2 into oxygen and water. Peroxidases are heme-containing enzymes as well and inactivate H 2 O 2 by reducing it to water. In addition to heme-containing catalases and peroxidases, nonheme varieties of these enzymes exist. Different cellular substrates can serve as electron donors for this reaction. Frequently, organisms use different isozymes, which are expressed simultaneously or under developmental-stage-and environment-specific conditions (26,35).
One good example for the employment of several catalases and their differential regulation during the life cycle is the filamentous fungus Aspergillus nidulans (15). A. nidulans is able to grow as vegetative hyphae but then undergoes two developmental programs. After 20 h of vegetative growth it can enter an asexual reproductive pathway in which it generates thousands of single-cell, haploid conidiospores. In addition, it is able to reproduce itself with very durable sexually derived ascospores (1). Both spore types are produced at or in special morphological structures, called conidiophores or cleistothecia, respectively. The conidiophore consists of four different cell types-a stalk, metulae, phialides, and conidia-and grows away from the agar surface into the air. The asexual developmental pathway is very well characterized at the molecular level (1) and is triggered by a central cascade of transcriptional activators (3,22). In an effort to characterize differentially expressed genes during asexual development, a catalase gene (catA) was discovered (20) that is transcriptionally and posttranscriptionally regulated, and the protein accumulated in conidiospores (19). Using the catA sequence, a second catalase, catB, was isolated (16). This gene is developmentally induced during conidiophore formation, but the transcript is almost absent in conidiospores. The catB expression, like that of catA, also responds to different stress conditions (6,16). Finally, Kawasaki and Aguirre, taking advantage of the genomic sequencing project at Cereon Genomics LLC (Cambridge, Mass.), identified a third catalase gene, designated catC (15). The protein resides in peroxisomes and is constitutively expressed. Interestingly, mutations in a single gene or in all catalase genes did not have any detectable vegetative or developmental phenotype, suggesting the presence of more isozymes. Indeed, in native polyacryl amide gels a fourth catalase activity, catD, was observed (15). We have identified the corresponding gene and found that it is a catalase-peroxidase. The expression of the gene is transcriptionally and translationally regulated upon carbon starvation and during sexual development. One important regulator is the transcription factor StuA.

RESULTS
Cloning of the catalase-peroxidase gene, cpeA. In an attempt to isolate a gene that is induced during sexual development, we purified laccase II. This enzyme was described as being specifically expressed in sexual structures such as Hülle cells and primordia (13,24). The protein was purified to apparent homogeneity, and three internal peptide sequences were determined, one of which showed sequence similarity to prokaryotic catalase-peroxidases. The internal peptide sequences were used to screen the partial genomic database at Cereon Genomics LLC, where we identified a DNA sequence (579 bp) whose translation product matched the peptides (Fig. 1). The DNA sequence information was used to isolate a corresponding cosmid from the pKBY library (FGSC). After subcloning of the cpeA gene as 3-kb PstI, 2.5-kb BamHI, and 5.5-kb EcoRV restriction fragmenta, we sequenced the locus on both strands (Fig. 1). cDNA was generated by reverse transcription-PCR with total RNA as a template for the reverse transcriptase reaction. Sequence comparison revealed no intron in the coding region. The sequence was translated into a 739-amino-acid (aa) polypeptide with a predicted molecular mass of 81.2 kDa. The polypeptide displayed, like one of the other peptides, a high degree of similarity to catalase-peroxidases of prokaryotic origin, with amino acid identities ranging from 54% to one of the archaebacterium, Halobacterium salinarum, to 64% of Streptomyces reticuli CpeB. CpeA is more distantly related to yeast cytochrome c peroxidase (Ccp1) and plant L-ascorbate peroxidases. The A. nidulans and Neurospora crassa (accession no. AF459787) sequences are the first two examples of complete sequences of catalase-peroxidases of eukaryotic origin (21). The two sequences share 69.3% identical amino acids. Alignment of CpeA (739 aa) with Saccharomyces cerevisiae Ccp1 (361 aa) showed that CpeA is similar to Ccp1 in the first and second halves of the protein (data not shown). This suggests a gene duplication event during evolution as suggested for the prokaryotic enzymes (32). Direct comparison of the first 439 aa of CpeA with the C terminus revealed 22% identical amino acids. If conserved amino acid changes were taken into consideration, the similarity went up to 40%. These values are comparable to 21% identical amino acids in the alignment of the two halves of the E. coli enzyme HPI.
Residues that have been shown to be essential for the catalytic activity of bacterial catalase-peroxidases and cytochrome c peroxidase are conserved in the A. nidulans enzyme. An amino acid triad, Arg-Trp-His, at the distal active site matches the positions R91, W94, and H95 in CpeA. A His proximal heme ligand aligns to H258, and a Trp residue shown to be important for peroxidase activity of Ccp1 is conserved in W309.
Upstream of the cpeA open reading frame we found a FIG. 1. Sequence of the catalase-peroxidase (cpeA) and the pyrroline-5-carboxylate reductase (pcrA) locus. (A) A nucleotide sequence of the depicted area was obtained from both strands by using a 3-kb PstI, a 2.5-kb BamHI, and a 5.5-kb EcoRV restriction fragment as a template. The derived amino acid sequences, obtained after removal of one intron in the pcrA gene, are denoted below the DNA sequence in the single-letter code. Since the two genes are encoded on opposite strands of the DNA, we indicated the direction of translation at the start codons with arrows. The intron border sequences of pcrA are indicated. The amino acids of CpeA involved in heme coordination are boxed. The peptide sequences obtained previously from purified CpeA protein are shaded. The sequence used for primer extension is underlined (dotted line). The putative binding sites for the regulators StuA (StRE) and CreA are indicated in the cpeA promoter region by dark and light gray boxes, respectively. The striped boxes indicate the location of four putative AreA binding sites. The determined transcription start site is labeled with an asterisk above the sequence. The two primers (prom2 and prom1) used for the amplification of the promoter with or without the StuA binding site are indicated by a line above the DNA sequence. The displayed sequence of the cpeA and the pcrA genes are available under the accession numbers AJ305225 and AJ313094. polypeptide encoded on the other strand (Fig. 1). After the removal of one predicted intron from the sequence, we deduced a protein of 284 aa with a calculated molecular mass of 29.2 kDa. The protein displays high homology (45.7% identical amino acids) to pyrroline-5-carboxylate-reductase from Pseudomonas aeruginosa (accession no. AE004476) and thus was named pcrA. This enzyme is involved in proline biosynthesis. Disruption of cpeA. Although CpeA was purified by using the laccase enzyme assay, the isolated gene encoded a catalaseperoxidase rather than a laccase. In order to answer the question whether the purified protein was a trifunctional enzyme or whether the two enzymes were copurified, we disrupted the cpeA open reading frame by using the nutritional marker gene argB. We transformed the wild-type strain RMSO11 with the linearized construct (pHPR12) and obtained three strains with the homologous integration event as shown in Fig. 2. Two strains (SMH1 and SMH4) were used for further analysis. To our surprise, protein extracts of the two disruptant strains still had laccase activity. To further characterize the strains, we analyzed catalase and peroxidase activities by using native polyacrylamide gel electrophoresis and subsequent activity stainings. We were able to detect one band in the peroxidase and the catalase assay, which were absent in the disruptant strain (Fig. 2). The other catalases-CatA, CatB, and CatCwere not resolved properly because of overloading of the gel. The catalase band due to CpeA had the highest mobility in the gel, a finding that is in agreement with the properties of CatD described before (15). These results strongly suggest that catalase-peroxidase copurified with laccase II. Interestingly, in N. crassa the purified fraction of catalase-peroxidase also contained laccase activity (W. Hansberg, unpublished data). This suggests that the purification protocol used in both filamentous fungi was not sufficient to separate the two proteins.
To analyze the biological function of the bifunctional enzyme, we compared a wild-type strain with SMH1 and SMH4 and found no significant differences with respect to hyphal growth, asexual spore formation, sexual spore production, or viability of the spores. However, we found an interesting regulation of gene and protein expression.
CpeA is expressed during early sexual development and upon carbon starvation. After the determination and analysis of the primary structure of the CpeA protein, we wanted to analyze the temporal expression pattern of the gene. We inoculated strain FGSCA4 onto agar plates, incubated the plates at 37°C for several days, and harvested the cultures after defined time periods. RNA was extracted and subjected to a Northern blot analysis (Fig. 3). Although no transcript was detectable in hyphae (vegetative), the signal increased when cells entered the sexual cycle (after 40 h).
Because catalases and peroxidases are required to cope with reactive oxygen species, we tested whether other growth conditions, in addition to sexual development, would induce transcription of cpeA. Mycelia were grown in liquid culture for 20 h at 37°C and then either exposed to an agar surface to induce development, further grown in submerged culture with or without glucose or nitrogen, grown in submerged culture with alternative carbon sources, or grown under oxidative stress conditions, under osmotic stress, or at high temperature (Fig. 3). After 3 h of growth under these conditions the mycelia were EcoRV-EcoRV fragment were cloned into pBluescript, and the argB marker was inserted as an EcoRI restriction fragment in between them. (B) Southern blot analysis to confirm the integration event. Genomic DNA of a wild type (WT) and a disruptant strain (SMH1) was digested with EcoRI and hybridized to the 5.3-kb XhoI-EcoRV fragment depicted in panel A. (C) Activity stain of peroxidase and catalase in a native 10% polyacrylamide gel. The wild type and the disruptant strain (SMH1) were induced for sexual development, and mycelia were harvested after 72 h of growth. The total protein (50 g) was separated in a native polyacrylamide gel, and activities were determined by staining as described earlier (34). The asterisk indicates the catalase activity not present in the diruptant strain. The other catalase activities visible above this band are found in both strains and indicate equal loading of protein. A total of 10 5 conidiospores were inoculated onto membranes and exposed onto a minimal medium agar plate. The plates were incubated at 37°C, and mycelia were harvested after 16 h (veg.), 24 h when asexual development was completed (asex.), and during sexual development at the time points indicated. RNA was isolated from the mycelia and hybridized to a cpeA-specific probe (upper panel). Then, 2 g of the same RNA preparation was loaded onto a gel and stained with ethidium bromide as loading controls for the gel. (B) Northern blot analysis of RNA isolated from mycelia after different treatments. Conidia of FGSCA4 were inoculated in complete liquid medium and incubated at 37°C for 20 h. The mycelium was harvested and divided into nine equal portions. One fraction was resuspended in minimal medium and incubated for 3 h before RNA isolation (lane "control"). One fraction was induced for development (lane "development") and then used for RNA isolation. The other seven samples were resuspended in minimal medium containing 0.5 mM H 2 O 2 -4% ethanol instead of 2% glucose-100 mM glucose plus 1 M sorbitol or 1 M NaCl into medium lacking any nitrogen or carbon source. Heat shock treatment was performed at 42°C. The treatments are indicated above the lanes. RNA was processed as described in panel A. (C) Northern blot analysis of RNA from mycelia induced for development and grown at a low glucose concentration (0.8%) in which no sexual development occurred. The time points of harvesting are indicated above the lanes. harvested, and the cpeA transcript levels were determined by Northern blot analysis. Besides the developmental induction after 40 h, we observed a strong signal when the mycelia were starved for carbon and a less-intense signal when the mycelia were starved for nitrogen. The repression of the gene under high-glucose conditions could be mediated through the carbon catabolite repressor CreA, for which we found several putative binding sites in the promoter region (Fig. 1). Induction upon nitrogen limitation could be achieved by the transcription factor AreA (Fig. 1). A slight induction of the expression of CpeA was also observed when ethanol was used as sole carbon source. A similar expression pattern was found for CatD (15).
To determine whether transcriptional activation during sexual development is due to carbon starvation of the mycelium, we grew A. nidulans in the presence of 0.8% glucose instead of 2% glucose. Under these conditions vegetative and asexual development occurs normally, but sexual development is not initiated (33). The cpeA transcript only accumulated transiently to a low level but did not reach the transcript levels observed during sexual development (Fig. 3).
Posttranscriptional regulation. After the analysis of gene expression at the transcriptional level we wanted to study the timing of protein appearance. To detect the polypeptide, we constructed a translational fusion with the GFP (SMS14) and analyzed the fusion protein in Western blot analyses with anti-GFP antibodies (Fig. 4). No specific band was detected in protein extracts obtained from starved hyphae, although cpeA transcript was induced to a high level. The results were confirmed when we microscopically inspected the hyphae after 3 and 6 h. Green fluorescence of the hyphae only appeared after 24 h of starvation (results not shown). This suggests a long delay between transcription and translation. The mechanism for this posttranscriptional control remains to be elucidated.
In analyzing the CpeA-GFP fusion protein, we observed that the protein is C terminally cleaved (Fig. 4). We observed, in addition to the protein band of ca. 100 kDa, a band of ca. 30 kDa. Since the CpeA protein was tagged at the C terminus, the small band must represent a small portion from the C terminus. The N-terminal part is not detectable in this strain. The small form was especially enriched in older cultures induced for sexual development. When Hülle cells were 1 day old (72 h of growth), only the high-molecular-mass band (100 kDa) appeared, whereas when the enzyme was detected in extracts from 2-day-old Hülle cells the smaller (30-kDa) product was observed. Whether this phenomenon can be explained by partial proteolysis or whether it is of functional importance cannot be decided yet. Interestingly, a processing of catalase-peroxidase was reported recently in Streptomyces reticuli, where the C-terminal part of the protein is necessary for manganese peroxidase activity (34).

CpeA is a cytoplasmic enzyme and localizes in Hülle cells.
To study the spatial distribution of the enzyme, we fused sGFP translationally to the carboxyl terminus of CpeA and expressed the fusion construct under the control of the natural cpeA promoter. We transformed pMS42 into SMS8 and performed Southern blot analysis to analyze the integration pattern of the construct. In two of the transformants the construct was integrated homologously at the cpeA locus, resulting in a gene duplication with the transgenic copy under the control of the natural promoter (results not shown). Since this situation represents the natural regulation status and should not cause misscheduled expression because of genomic integration someplace in the genome, we selected one of these strains (SMS11) for further analysis. Microscopic inspection under fluorescence conditions revealed that Hülle cells were brightly stained (Fig. 5), whereas primordia were weakly stained and  (9). cpeA expression is activated through stuA. In order to study the regulatory circuits leading to a developmental upregulation of cpeA transcription, we tested whether known regulators affect the expression of the gene. First, the start of transcription was determined by primer extension 52 bp upstream from the predicted translational start (Fig. 1). Analysis of the DNA sequence upstream of the transcription start site revealed binding sites for the regulators BrlA, AbaA, and StuA (the StuA binding site is indicated in Fig. 1). Therefore, we have chosen strains with mutations in brlA, abaA, or stuA; analyzed the induction of cpeA during sexual differentiation; and found that only in a stuA mutant was developmental induction greatly reduced (Fig. 6). Since cpeA and pcrA share the promoter region and the StuA binding site is located within the open reading frame of pcrA, we analyzed the transcription of the upstream gene as well. In the wild type, pcrA is downregulated at the time when cpeA becomes induced. The downregulation of pcrA was unaffected in the stuA1 mutant (Fig. 6). To further prove that StuA is directly responsible for the observed induction of cpeA, we used a strain in which the promoter of stuA was replaced by the alcA promoter. The latter promoter is repressed in the presence of glucose and highly induced by ethanol or threonine. This allows high expression of StuA in the absence of developmental processes, namely, in submerged culture. This time and spatially mis-scheduled expression of stuA should lead to an induction of target genes if no other development-specific transcription factor is required (the alcA fusion strains were kindly provided by B. Miller, Moscow, Idaho). In addition, we tested for the stimulation of cpeA expression upon induction of BrlA or AbaA by using the same experimental approach. We grew the corresponding strains in the presence of glucose and shifted the medium after 20 h to medium containing ethanol for 1, 2, and 3 h. The mycelia were harvested and processed for Northern blot analysis. As already observed during expression analyses, cpeA was induced in wildtype cells due to ethanol as carbon source (Fig. 3). In these shift experiments, we observed that transcription was induced after 1 h; the transcript level then decreased (by 2 h), and after 3 h it increased again. The same pattern of transcription was observed in strains that expressed brlA or abaA under the control of the alcA promoter, indicating that these two regulators are not involved in cpeA activation. However, in the strain with the alcA::stuA construct, the transcript level remained high at all time points after induction of the alcA promoter. This suggests that StuA induces the expression of cpeA (Fig. 6).
In the cpeA promoter region, several potential StuA binding sites with the core sequence CGCG were detected. One of them (indicated in Fig. 1

) matches the full consensus of the StRE [(A/T)CGCG(T/A)N(A/C)] (8).
To further prove the effect of StuA on cpeA expression, we deleted this putative StuA binding site from the cpeA promoter in the cpeA::gfp fusion construct and monitored the expression by Northern blot and fluorescence microscopy ( Fig. 6 and 7). When we used the construct with the StuA binding site (SMS14, with a single integration of pMS41), we detected an upregulation of the transcript during development and upon starvation. Hülle cells showed strong fluorescence in their cytoplasm, and we observed weak fluorescence in the dikaryotic mycelium of the fruiting bodies. In contrast, the promoter without the StuA binding site (SMS16, with a single integration of pMS42) did not support developmental induction, whereas the response to carbon starvation was unaltered. The CpeA-GFP fusion protein was no longer detectable in Hülle cells, but the expression in the dikaryotic mycelium remained. This shows that StuA is responsible for the tissue-specific induction of CpeA.

DISCUSSION
In this study we have analyzed catalase-peroxidase of A. nidulans and found that the gene is expressed upon carbon starvation and is highly induced during sexual development in Hülle cells. We tested several potential regulators for an effect on cpeA expression. Among those were nsdD and veA, with a The stuA gene was isolated by complementation of a developmental mutant that is blocked in asexual development, unable to enter the sexual cycle, and does not even produce Hülle cells (17). StuA belongs to a group of regulators of fungal morphology sharing a common DNAbinding motif named the APSES domain, the term being derived from the currently known members of the group: ASM-1, Phd1, StuA, Efg1, and Sok2 (4). The APSES motif has structural similarity to the DNA-binding domain of eukaryotic basic helix-loop-helix (bHLH) proteins (8), which form homo-and heterodimers mediated by the HLH domain (2). The StuA protein binds to promoter elements with the consensus sequence (A/T)CGCG(T/A)N(A/C) and has been shown to act as a repressor of the central regulators of asexual development abaA and brlA to allow their correct temporal and spatial expression (8,30). Since we found that StuA enhances the expression of cpeA during sexual development, it can be concluded that the regulator is able to mediate both negative and positive transcriptional control. One possible explanation for this dual function is to assume interaction of StuA with different partners via HLH domains to form distinct heterodimers during asexual and sexual development, which then could act as repressors or activators of transcription, respectively. Similarly, a dual function as repressor and activator of transcription has been proposed for the Candida albicans homologue of StuA, Efg1, which is essential for hyphal development in the fungal pathogen (27). Formation of multiple dimer combinations of bHLH proteins is a well-known mechanism for regulation of differential gene expression in mammals (2). Additional research is needed to identify potential dimerization partners of StuA and to unravel their different contribution to the regulation of the morphogenetic pathways in A. nidulans.
Role of catalase-peroxidase during sexual development. Catalases and peroxidases are involved in the detoxification of harmful oxygen species. The complex regulation of the expression of the three catalases in A. nidulans suggests distinct enzymatic properties that are required at specific stages of the life cycle. Whereas CatA is mainly expressed in asexual and sexual spores, CatB is found in the hyphae and is induced during asexual sporulation but is absent from conidia and CatC is expressed in hyphae (15). Most likely, catalase-peroxidase serves a similar function in coping with deleterious oxygen molecules, as do the other catalases. Since the initial stages of FIG. 6. Transcriptional activation of cpeA is regulated through StuA during sexual development. (A) RNA was isolated from FGSCA4 and a stuA1 mutant strain (GO256) at the time points indicated above the lanes. RNA was hybridized to cpeA (upper panel)-and pcrA (middle panel)-specific probes. rRNA was stained on the membrane with methylene blue as a loading control (lower panel). (B) Forced expression of stuA, brlA, and abaA was achieved by growth of the strains for 20 h on glucose medium (repressing conditions) and then shifted to ethanol-containing medium for the times indicated above the panels. RNA isolated from wild-type strain FGSCA4 and the three transgenic strains UI132 (alcA::stuA), TPM1 (alcA::abaA), TTA292 (alcA::brlA) was processed for a Northern blot. The membrane was hybridized to a cpeA specific probe (upper 4 panels) and to a stuA, brlA and an abaA specific probe (lower 3 panels). (C) The transgenic strain SMS14 (cpeA::gfp) and SMS16 [cpeA(⌬StuA binding sites)::gfp] were analyzed for cpeA-gfp expression after the induction of development. As a probe for the Northern blot, we used gfp (upper panels) or cpeA (lower panels). rRNA staining was used as loading control. (D) Strains SMS14 and SMS16 were also analyzed for CpeA-GFP expression upon carbon starvation (lower panels). gfp or cpeA were used as probes as indicated. Another attractive possibility for a role of catalase-peroxidase during development comes from the observation that in N. crassa a hyperoxidant state was detected at the start of different morphogenic transitions (12,29). Therefore, CpeA could have an important function in coping with this dangerous state. It has even been postulated that this hyperoxidant state triggers differentiation (11). Likewise, in N. crassa, catalaseperoxidase was isolated from conidiating cultures and has been detected in cells in the stationary phase when they showed increased vacuolization and begin lysing (21).
More evidence for an association of sexual development with oxidative stress comes from studies in Podospora anserina. It was observed that the number of peroxisomes, a cellular compartment, which harbors enzymes to cope with oxidative stress, increases dramatically during ascus formation (5). Mutation of a gene, car1, involved in the biogenesis of these organelles resulted in a karyogamy defect and thus a block in sexual development. Since Podospora anserina and some Aspergillus species produce fruiting bodies but lack Hülle cells, the Hülle cells in A. nidulans could only be one strategy to cope with oxidative stress. This is supported by the fact that loss-offunction of the two enzyme activities did not lead to any strong developmental phenotype. These results suggest that a concerted action of different enzymes is employed to detoxify reactive oxygen species in different cell types and that successful protection can be achieved by different and overlapping functions.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Induction of vasculogenesis in breast cancer models

Recently, there have been reports of postnatal vasculogenesis in cases of ischaemia models. The aim of the present study is to provide evidence of postnatal vasculogenesis in breast-cancer–bearing mice. Based on cell surface antigen expression, we isolated endothelial precursor cells from bone marrow, peripheral blood and tumour-infiltrating cells from mice that had received six human breast cancer xenografts. In all three areas (bone marrow, peripheral blood and tumour-infiltrating cells), endothelial precursor cell population was elevated in all transplanted mice. Differentiation and migration activities of endothelial precursor cells were measured by comparing levels of the endothelial precursor cell maturation markers Flk-1, Flt-1, Tie2, VE-cadherin and CD31 among these three areas. The endothelial precursor cell population was 14% or greater in the gated lymphocyte-size fraction of the inflammatory breast cancer xenograft named WIBC-9, which exhibits a hypervascular structure and de novo formation of vascular channels, namely vasculogenic mimicry (Shirakawa et al, 2001). In vitro, bone marrow-derived endothelial precursor cells from four human breast cancer xenografts proliferated and formed multiple clusters of spindle-shaped attaching cells on a vitronectin-coated dish. The attaching cells, which incorporated DiI-labelled acetylated low-density lipoprotein (DiI-acLDL) and were negative for Mac-1. The putative bone marrow derived endothelial precursor cell subset, which was double positive of CD34 and Flk-1, and comparative bone marrow derived CD34 positive with Flk-1 negative subset were cultured. The former subset incorporated DiI-acLDL and were integrated with HUVECs. Furthermore, they demonstrated significantly higher levels of murine vascular endothelial growth factor and interleukin-8 in culture supernatant on time course by enzyme-linked immunosorbent assay. These findings constitute direct evidence that breast cancer induces postnatal vasculogenesis in vivo. British Journal of Cancer (2002) 87, 1454–1461. doi:10.1038/sj.bjc.6600610 www.bjcancer.com © 2002 Cancer Research UK

Postnatal neovascularisation is thought to result exclusively from proliferation and remodelling of fully differentiated ECs derived from pre-existing blood vessels (Holash et al, 1999). This adult paradigm, referred to as angiogenesis, contrasts with vasculogenesis, the term applied to the formation of embryonic blood vessels from endothelial precursor cells (EPCs). Recently, however, EPCs have been isolated from adult peripheral blood (PB) and umbilical cord blood, and these cells attach themselves to vitronectin-coated dishes (Asahara et al, 1997;Isner and Asahara, 1999;Takahashi et al, 1999;Kalka et al, 2000;Murohara et al, 2000). Augmentation of transfused EPCs has been observed in ischaemic tissue, and participation of transfused EPCs in neovascular formation has been observed in mature animal models. Previously, we reported the establishment of a new human IBC xenograft (WIBC-9) that exhibits angiogenesis and de novo formation of vascular channels, vasculogenic mimicry (Shirakawa et al, 2001). WIBC-9 overexpresses human (h) angiogenic factors , vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 (Ang-1)) and murine (m) angiogenic factors (integrin avb3, flt-1, tie-2, VEGF, Ang 2), compared with 3 non-IBC xeno-grafts, at the mRNA and/or protein level. Previously, by blocking the VEGF-Flt-1 and Angiopoietin 1,2-Tie2 pathways in IBC, we achieved a ratio of tumour growth inhibition of 99% or greater and demonstrated marked anti-angiogenic and putative antivasculogenic effects (Shirakawa et al, 2002). In the present study, we tested our hypothesis that human breast cancer lines (specifically, WIBC-9) can induce proliferation, of ECs and EPCs in animal models.

Xenografts
WIBC-9 and the established non-IBC xenografts MC-2, MC-5 and MC-18 were maintained as described elsewhere (Shirakawa et al, 2001). SK-BR3, MCF-7, SK-BR3/hVEGF and MCF-7/ hVEGF cells were injected subcutaneously into the second mammary fat pads of athymic, female, ovariectomised 4-weekold BALBc nu/nu mice (10 7 cells 100 ml 71 serum-free culture medium). To evaluate EPCs and hVEGF concentration in peripheral blood, retro-orbital bleeding into capillary tubes was undertaken from tumour-bearing mice (n=5 per study group) when the tumour became 10 mm in maximum diameter after transplant. Femurs were also undertaken from these mice to extract bone marrow. All animal experiments have been carried out with ethical committee approval. The ethical guidelines that were followed meet the standards required by UKCCCR guidelines (Workman et al, 1998). WIBC-9 and the established non-IBC xenografts MC-2, MC-5 and MC-18 were maintained as described elsewhere (Shirakawa et al, 2001).
The animals received a weekly percutaneous administration of 100 mg of 17b estradiol (Sigma, Saint Louis, MO, USA) in 10 ml of ethanol, in order to obtain tumours with a volume of 1000 mm 3 .

Mobilisation of ECs and EPCs by recombinant human GM -CSF (rhGM -CSF) and rhVEGF
rhGM -CSF (molecular weight, 14 kD) and rhVEGF (molecular weight, 38.2 kD), in powdered form (Sigma), were dissolved in phosphate-buffered saline (PBS). To determine the mobilisation activity of GM -CSF and VEGF on ECs and EPCs, 50 ng of rhGM -CSF or 10 ng of rhVEGF was injected into BALB/c nude mice (Clea Japan, Tokyo, Japan) intraperitoneally as in vivo controls.
Preparation of cultured conditioned medium and determination of hVEGF concentration by ELISA SK-BR3, SK-BR3/hVEGF, MCF-7 and MCF-7/hVEGF cells (1610 6 cells well 71 ) were incubated for 2 days at 378C, and the supernatants were then collected and stored at 7808C until used. Concentration of hVEGF was measured by ELISA in 100 ml samples of supernatant, using immunoassay kits (Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan). Each assay was performed in triplicate.

EPC culture
On day 1 of culture, BM-or PB-derived EPCs were cultured in Stem pro (GIBCO BRL, Grand Island, NY, USA). Floating MNCs and sorted mCD34-positive MNCs were cultured overnight on non-coated plastic plates, and 5610 5 cells were then transferred to murine fibronectin-coated plastic plates (GIBCO BRL) for culturing. From day 2 of culture, EPCs were cultured in SFM (GIBCO BRL) supplemented with 20% FBS and bovine pituitary extract. On days 4 and 13 of culture, AT cells and clusters were Experimental Therapeutics  Figure 1 Flow cytometry (quantitation of EPCs and ECs). In WIBC-9 xenografted mice, the populations of EPCs were markedly elevated in MNCs derived from BM, PB and TI cells. The following MNC subtypes were clearly visualised: CD34 + /Flk-1 + , CD34 + /Flt-1 + , CD34 + /Tie2 + and CD34 + /VE-cadherin + . In WIBC-9 mice, the population of cells that were CD45 7 /TER 7 /Mac-1 7 /CD34 + was approximately 10%, almost equal to the population of cells that were CD34 + /Flk-1 + MNCs. The EC population (CD34 + /CD-31 + MNCs) was also high in WIBC-9 mice, although not as high as the EPC population. EC populations of MC-5 xenografted mice were higher than those of the controls.

Immunocytochemistry
Spindle-shaped AT cells observed at 14 days of culture were subjected to immunocytochemistry to analyse the expression of Mac-1-FITC (Pharmingen).

Cellular uptake of acetylated LDL
We investigated Ac-LDL uptake (a process characteristic of endothelial lineage) into AT cells (Asahara et al, 1997). AT cells cultured on fibronectin were incubated in medium containing 10 mg ml 71 DiI-labelled Ac-LDL (DiI-Ac-LDL; Molecular Probes, Eugene, OR, USA) for 24 h at 378C. Cells were then examined under a fluorescence microscope.

Culture of EC and EPC on HUVEC monolayer
BM-derived MNCs from WIBC-9 xenografted mice that are mCD34 + and mFlt-1 + (sorted by FACS), were cultured in Stem pro (GIBCO BRL, Grand Island, NY, USA) for 13 days and incubated in the medium containing 10 mg ml 71 DiI-labelled Ac-LDL (DiI-Ac-LDL; Molecular Probes, Eugene, OR, USA) for 24 h at 378C. These cells were seeded on HUVEC monolayer cultured on Growth Factor Reduced Matrigel matrix (Becton Dickinson Labware, Bedford, MA) on day 5.

Statistical analysis
All data are expressed as the mean+s.d. StatView computer software (ATMS Co., Tokyo, Japan) was used for the statistical analysis of differences in results of MTT, migration assay and EPC population between groups. Two-sided P50.05 and P50.01 were considered to indicate statistical significance.

RESULTS
Flow cytometry (quantitation of EPCs) ( Table 1 and Figures 1 and 2) The population of EPCs was markedly elevated in all three sources of MNCs (BM, PB and TI) in WIBC-9 xenografted mice. The MNC subtypes that were clearly visualised were CD34-positive (CD34 + )/Flk-1 + , CD34 + /Flt-1 + , CD34 + /Tie2 + and CD34 + /VE-cadherin + . It should be noted that, in WIBC-9 mice, the population of cells that were CD45-negative (CD45 7 )/ TER119 7 /Mac-1 7 /CD34 + (considered part of the EPC population) was around 10%, which is almost equal to the number of CD34 + /Flk-1 + MNCs in BM and PB. The EC population (CD34 + /CD31 + MNCs) was also prominent in WIBC-9 mice. The real number of MNCs in PB was also elevated in WIBC-9, MCF-7/hVEGF, and rhGM -CSF mice. EPC and EC populations were slightly elevated in MC-5. These results are shown in  Figure 3 (A) AT cell population in the xenografts. AT cells, including ECs were counted. In WIBC-9 samples, the AT cell population was high. AT populations in other cancer lines were higher than in the control, a finding consistent with the size of the CD34 + /Flk-1 + MNC population. (B) BM-derived EPC morphogenesis on murine fibronectin. On day 4 after plating CD34 + floating MNCs, spindle-shaped AT cells and cluster formation were evident in WIBC-9 samples. On day 13, all AT cells were found to uptake DiI-labelled acLDL. Immunocytochemistry revealed that all cells were Mac-1 -negative. Each scale bar is 50 mm. AT cell population in samples from xenografted mice ( Figure 3A) In WIBC-9 samples, AT cells comprised markedly high percentages of total cells (20% or greater). In other lines, AT cell populations were higher than that of the control, a finding consistent with the observed CD34 + /Flk-1 + populations.
BM-derived EPC morphogenesis on murine fibronectin ( Figure 3B) On day 4 after plating the CD34 + floating MNCs, spindle-shaped AT cells and cluster formation were clearly visible in WIBC-9 samples. On day 13, WIBC-9 AT cells were found to uptake DiIlabelled acLDL. Immunocytochemistry revealed that these cells were negative for Mac-1, indicating that they were not of a monocyte lineage.

Culture of EC and EPC on HUVEC monolayer (Figure 4)
BM-derived MNCs that are mCD34 + and mFlt-1 + incorporated DiI-labelled acLDL and were integrated in a HUVEC monolayer (forming capillary networks) implying their maturation to ECs.
The production of angiogenic factors by putative EPCs (Figure 5) BM derived CD34 + Flk-1 + MNC subset and CD34 + Flk-1 7 MNC subset produced mVEGF and mIL-8 on time course. The former subset demonstrated significantly higher levels of murine VEGF and IL-8 in culture supernatant on time course by ELISA.

DISCUSSION
Neovascularisation encompasses both angiogenesis and vasculogenesis. In angiogenesis (the classic paradigm for new vessel growth), mature, differentiated ECs break free from their basement membrane, migrate and proliferate to form branches of parental vessels. The fact that tumours induce angiogenesis indicates that tumour development and metastasis are dependent upon neovascularisation, and suggests that this relationship involves angiogenic growth factors that are specific to neoplasms (Folkman and Klagsbrun, 1987;Kendall and Thomas, 1993;O'Reilly et al, 1997;Goldman et al, 1998;Tanaka et al, 1998;Ferrara and Alitalo, 1999;Mori et al, 2000). Although the therapeutic potential of anti-angiogenic factors is reportedly promising, their antiangiogenic mechanisms (inhibition of migration, proliferation and tube formation) have not been well characterised. Vasculogenesis involves participation of BM-derived EPCs that circulate to sites of neovascularisation, where they differentiate into mature ECs (Asahara et al, 1997Isner and Asahara, 1999;Kalka et al, 2000;Murohara et al, 2000). The results of the present study indicate that six human breast cancer lines, including the IBC line WIBC-9, induce postnatal EPC kinetics as well as EC kinetics. The chemotactic modulation of proliferation and migration of EPCs that we observed was mainly the result of VEGF mobilisation, a finding consistent with those of a previous study (Asahara et al, 1997). MCF-7/hVEGF and WIBC-9 showed markedly enhanced growth on mice and higher microvascular density by histology compared with MCF-7 in this series study (Shirakawa et al, 2001) as previously described (Lewin et al, 1999). This enhanced growth may be a result of contribution of endothelial precursors induced by angiogenic factor such as VEGF (de Bont et al, 2001). Moreover, MNCs which contain haematopoietic precursors as well as EPCs in peripheral blood markedly increased in WIBC-9 and MCF7/hVEGF xenografted mice. This may be due to the mobilisation of the common precursors for EPCs and haematopoietic cells residing in bone marrow led by the elevation of plasma levels of factors such as VEGF secreted by cancer cells (Hattori et al, 2001). Thus, our results support the notion that BM-derived precursors provide a sufficient source of ECs to enhance the growth of breast cancer in vivo. In vitro, the CD34 + floating MNCs on day 4 after plating, spindle-shaped AT cells and cluster formation were clearly visible in WIBC-9 samples. Although, we have already reported the TI EPCs in WIBC-9 xenografted mice (Shirakawa et al, 2002), the population of EPCs was markedly elevated in all three sources of MNCs (BM, PB and TI) in WIBC-9 among the xenografted mice. These facts possibly show the induction of vasculogenesis especially in WIBC-9. On day 13, AT cells from WIBC-9 were found to uptake DiI-labelled acLDL. Immunocytochemistry revealed that these cells were negative for Mac-1, indicating that they were not of a monocyte lineage. These facts possibly show the maturation from EPC to EC in vitro culture. In WIBC-9, human angiogenic factors (hAng1, hVEGF, hbFGF) and murine angiogenic factors (mflt-1, m integrin b3, mVEGF, and mCD31), were expressed at higher levels than they were in the three non-IBC xenografts. However, because our results indicate that the human breast cancer lines MC-2, MC-5 and MC-18 secrete low levels of hVEGF, modulation by accessory molecules constitutes a cytokine network, and autocrine or paracrine secre-Experimental Therapeutics Figure 4 (A) HUVECs and BM-derived EPCs (mCD34 + and mFlt-1 + ) formed capillary networks. (B) BM-derived EPCs, which incorporated DiI-labelled acLDL, were integrated with HUVECs. Phase contrast photomicrographs showed capillary networks. Each scale bar is 10 mm.
tion of angiogenic factors is likely to be associated with modulation of EPC recruitment (Shirakawa et al, 2001). The VEGF family receptor, mflt-1 and the Ang receptor, hTie-1, and hTie-2, mtie-2 was expressed at a higher level in WIBC-9, but mflk-1 and mflt-4 Experimental Therapeutics   Figure 5 BM derived CD34 + +Flk-1 + MNC subset and CD34 + +Flk-1 7 MNC subset (1610 5 cells well 71 ) were sorted by using FACS (n=5) and incubated for 14 days at 378C, and the supernatants were collected every 12 h and stored at 7808C until used. Concentrations of mVEGF and mIL-8 were measured by ELISA in 100 ml samples of supernatant, using Immunoassay Kits (Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan). Each assay was performed in triplicate.
were not detected in WIBC-9. Expression of hFlt-1 and KDR was detected in all xenografts. The cytokine, hIL-1b was only detected in WIBC-9, and a higher expression of hIL-8 was detected in WIBC-9. The adhesion molecule, h integrin b3, m integrin b3 and m integrin av were detected at higher levels in WIBC-9 as previously described (Shirakawa et al, 2001). BM-, PB-and TI-derived murine EPCs and ECs from human breast cancer-bearing mice were found to exhibit a specific pattern of cell surface antigen expression; i.e., double positive for mCD34 and mFlk-1. The subtypes, which may show the maturation of EPC, detected were mCD34 + /mFlk-1 + , mCD34 + /mFlt-1 + , mCD34 + /mTie-2 + , mCD34 + /mVE-cadherin + and mCD34 + / mCD31 + . In this model, we detected both EPCs and ECs, or their subtypes (maturation), among BM-, PB-and TI-derived cells. The data of multiple markers analysis, which are Flk-1, Flt-1, Tie-2, VEcadherin, and CD31 by using tri-color FACS, indicated that the populations of CD34 + +FLK1 + in the bone marrow and peripheral blood in WIBC-9, included a large number of FLT1+(98.9%/ 97.5%), and TIE2+(71.8%/64.2%) but include small number of VE-cadherin (23.2%/30.6%) and CD31+(20.9%/20.9%). Induced EPCs and ECs were clearly shown to be non-malignant cells, and May-Gimsa staining revealed that they were of the same phenotype as rhVEGF-and rhGM -CSF -mobilised EPCs and ECs (data not shown). Surprisingly, induced EPC populations which were positive of mCD34 and mFlk-1, showed the production of mVEGF and mIL-8 in culture supernatant on time course. This result implies autocrine regulation of proliferation of these precursors in vivo, and coincides with the recent report of the regulation of haematopoietic stem cells (Gerber et al, 2002). In our model, this autocrine cascade may play an important role of the induction of vasculogenesis and the tumour growth.
The chemotactic expansion of EPCs derived from PB or cord blood and transplantation of these EPCs have previously been reported (Asahara et al, 1997Isner and Asahara, 1999;Takahashi et al, 1999;Kalka et al, 2000;Murohara et al, 2000); transplanted EPCs were found to significantly proliferate in ischaemic foci and differentiate in situ through a process of vasculogenesis. In the present study, we demonstrated that breast cancer-bearing mice exhibit significant expansion of precursors including EPCs and ECs; specifically, maturation and proliferation of these cells in tumours was clearly evident.