Biochemical, Molecular, and Genetic Mechanisms

Biochemical, Molecular, and Genetic Mechanisms

Tp53-Associated Growth Arrest and DNA Damage Repair Gene Expression Is Attenuated in Mammary Epithelial Cells of Rats Fed Whey Proteins1 Bhuvanesh Dave, Renea R. Eason, Yan Geng, Ying Su, Thomas M. Badger, and Rosalia C. M. Simmen2 Arkansas Children’s Nutrition Center and Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72202

KEY WORDS: mammary gland Tp53 DNA damage monocyte chemoattractant protein-1 whey proteins

Breast carcinoma, the most prevalent cancer and the second leading cause of malignancy-related deaths in women in the Western hemisphere, is associated with hitherto unknown etiology (1,2). Many questions on the progression of this disease from benign dysplasia to tumors with metastasis remain unexplored; however, there is increasing acceptance, based on epidemiologic studies, that it has its origins during early development, which can be influenced by nutrition (3,4). Studies using rat and mouse models of carcinogenesis have provided support for this hypothesis (5–7). A unifying mechanism(s) for dietary control of mammary carcinogenesis remains undefined; nevertheless, the potential contribution of altered expression of several major oncogenes (c-myc, Her-2/neu) and tumor suppressor genes [phosphatase and tensin homolog deleted in chromosome 10 (PTEN),3

Tp53, breast cancer 1 gene (BRCA1)] to this phenomenon has been broadly proposed (8–10). The tumor suppressor Tp53 is considered to be one of the most important molecular players in the pathogenesis of all types of cancers. It is mutated or deleted in $50% of human cancers (11,12), and mice null for Tp53 develop tumors with high frequency (13). Loss of Tp53 function underlies decreased genomic stability and is associated with defects in DNA damage repair, loss of cell cycle control, and deregulated activation of apoptosis (12–14). In response to DNA damage signals, Tp53 is phosphorylated by a wide variety of protein kinases, leading to its activation, accumulation in the nucleus, and regulation of transcription of target genes (14,15). Direct gene targets of Tp53 include those involved in apoptosis, cell cycle arrest, and DNA repair (16–18). The reciprocal cooperation of Tp53 with other tumor suppressors (e.g., PTEN, BRCA1) to facilitate their cell growth and transformation activities has further strengthened its critical role as a ‘‘gatekeeper’’ tumor suppressor (12, 19,20). Regulation of Tp53 expression and/or activity, which are controlled at the levels of transcription, post-transcription, and protein stability, (21,22) may constitute a relevant mechanism(s) for the tumor protective effects of dietary factors. Whey proteins, by-products of the manufacture of cheese and curd, were shown to be tumor protective in animal models (7), and in limited clinical trials of cancer patients (23,24). These proteins exhibit potent antioxidant activities by inducing

1 Funds for this research were provided by the U.S. Department of AgricultureCRIS-6251-5100-005-02S. 2 To whom correspondence should be addressed. E-mail: simmenrosalia@ uams.edu. 3 Abbreviations used: BRCA1, breast cancer 1 gene; CAS, casein; CHOP, C/ EBP-homologous protein; DMBA, 7,12-dimethylbenz[a]anthracene; FBS, fetal bovine serum; GADD, growth arrest and DNA damage; GSH, glutathione; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; Mgmt, O(6)-methylguanine-DNA-methyltransferase; PND, postnatal day; PTEN, phosphatase and tensin homolog deleted in chromosome 10; qPCR, quantitative RT-PCR; Sesn, Sestrin; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling; wip1; wild-type p53-induced phosphatase 1; WPH, whey protein hydrolysate.

0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 14 November 2005. Initial review completed 12 December 2005. Revision accepted 17 February 2006. 1156

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ABSTRACT Dietary protection from mammary cancer is likely coordinated through multiple signaling pathways, based on the known heterogeneity of the disease and the distinct origins of mammary tumor cells. The present study examined the modulatory effects of dietary intake of whey protein hydrolysate (WPH) relative to casein (CAS), on mammary epithelial cell resistance to endogenous DNA damage using Tp53 gene expression and signaling as a readout, and on systemic proapoptotic and immune surveillance activity, in young adult female Sprague-Dawley rats. Rats were fed AIN-93G diets made with CAS or WPH as the sole protein source beginning at gestation d 4. At postnatal day (PND) 50, mammary glands of rats fed WPH had lower levels of activated Tp53 and p38 mitogen-activated protein kinase proteins, and reduced transcript levels for Tp53-associated DNA damage repair, growth arrest, and proapoptotic genes than those of CAS-fed rats. Serum from WPH-fed rats had greater apoptotic activity in MCF-7 tumor cells than that from rats fed CAS. Serum levels of monocyte chemoattractant protein (MCP)-1 were higher in WPH- than in CASfed rats. MCF-7 cells treated with CAS serum 1 recombinant rat MCP-1 had apoptotic activity and Tp53 and p21 gene expression levels comparable to those treated with WPH serum or recombinant MCP-1. Results indicate that mammary glands of rats fed a WPH diet are more protected from endogenous DNA damage than are those of CAS-fed rats, and identify MCP-1 as a potential serum biomarker for the positive effects of healthy diets. J. Nutr. 136: 1156–1160, 2006.

DIETARY WHEY PROTEINS AND MAMMARY Tp53

MATERIALS AND METHODS Rats, diet and tissue collection. Animal procedures were approved by the University of Arkansas for Medical Sciences Animal Care and Use Committee. Time-mated Sprague-Dawley rats were obtained from Charles Rivers Laboratories and were housed individually in polycarbonate cages under conditions of 248C, 40% humidity, and a 12-h light:dark cycle. Rats at gestation d 4 were randomly assigned to 1 of 2 semipurified isocaloric diets made according to the AIN-93G formulation (27), except that corn oil was substituted for soybean oil and the sole protein sources were either casein (CAS; New Zealand Milk Products) or a partial hydrolysate of whey (WPH; New Zealand Milk Products). Rats from both diet groups were allowed to consume food and water ad libitum and did not differ in food intake, consistent with previous studies (7). At delivery, pups from dams of the same diet groups were pooled and 10 pups (5 per sex) were randomly assigned to each dam for suckling. Female pups were weaned at postnatal day (PND) 21 to the same diets as their dams and were fed the same diets throughout the study. Male pups were used in unrelated studies. At PND 50, female pups (10/diet group) were killed by injection with 100 mg Nembutol/kg body weight (Avent Laboratories), followed by decapitation, and the abdominal mammary gland (number 4) pair was removed. The left gland was frozen at 2808C for protein analysis, and the right gland was homogenized in TriZol for RNA extraction and analysis of gene expression. Nuclear extracts and Western blotting. Nuclear extracts were prepared from frozen mammary tissues using an extraction system (NE-Per; Pierce Biotechnology). Western blot analysis followed previously described protocols (7). The following primary antibodies were used under conditions recommended by the manufacturers: anti-Tp53 (Cell Signaling Technology); anti-Phospho-Tp53Ser15 (P-Tp53Ser15, Cell Signaling Technology); anti-p38 mitogen-activated protein kinase (MAPK; Santa Cruz Biotechnology), anti-phospho-p38 MAPK (Santa Cruz Biotechnology), and lamin B1 (Abcam). Data were normalized to the signals obtained with the lamin B1 antibody. RNA extraction and quantitative real-time RT-PCR (qPCR). Procedures for RNA isolation, cDNA synthesis, and primer design were as previously described (7). The primer sequences for the rat genes were as follows (Forward and Reverse, respectively): 1) wildtype p53-induced phosphatase 1 (wip1; 59-TAT TAG CAG CAC

CAT CTG TTA TTG AAA T-39; 59-CCC AGA CCA GTG GCA TTA GG-39); 2) O(6)-methylguanine-DNA-methyltransferase (Mgmt) (59-GTG AAA TTC GGA GAA ATG GTT TCT TA-39; 59-ACT GGA TTG CTC CTC ATC GC-39); 3) growth arrest and DNA damage (GADD)153/C/EBP-homologous protein (CHOP) (59- GAG TCT CTG CCT TTC GCC TTT-39; 59- GGT GCC CCC AAT TTC ATC T-39); 4) Sestrin (Sesn) 1 (59-TGC TTT GGG TCG TTT GGA TAA-39; 59-CCC GTC CAT CTG CAG TAG GT-39); and 5) BRCA1 (59-GAA GAG TAG CAT CAG TGA CTG CAA TAA-39; 59-TGT GAG GAG AAC AGC TGC C-39). The primer pairs for p21, PTEN, Tp53, and Bax, and the amplification reaction protocols were described previously (7,11). The target mRNA expression was normalized to that of 18S rRNA and expressed as arbitrary units (means 6 SEM). Cell culture, treatments, and assays. Human MCF-7 mammary tumor cells were obtained from BD Biosciences and cultured in DMEM containing 10% (v:v) fetal bovine serum (FBS), and 1% antibiotic: antimycotic solution (GIBCO) in 5% CO2:95% air at 378C. Human MCF-10A nontumor mammary cells (ATCC) were grown in DMEMF12 containing 5% (v:v) horse serum, insulin (10mg/L), epidermal growth factor (20mg/L), cholera toxin (100mg/L), hydrocortisone (0.5 mg/L), and 1% antibiotic:antimycotic solution. For terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells were seeded at a density of 1.2 3 105 cells/well on immunofluorescence chamber slides (Fisher Scientific). After 24 h of incubation, the cells were washed with PBS and incubated in fresh DMEM containing 0.5% FBS. Sera pooled from PND 50 rats fed CAS (n 5 10) or WPH (n 5 10) were added at 1% final concentration. Treated cells were processed 24 h later for detection of apoptotic cells using fluorescein-labeled TdT reagent (Oncogene) (7). Labeled nuclei were counted in 3 separate fields (100X magnification) containing 200–300 cells each, using an Olympus IX-71 microscope with a standard fluorescence filter. Data are presented as the percentage of labeled nuclei from the total number of cells counted. Cell viability of MCF-7 cells was assessed using the MTT assay as described by the manufacturer (Promega). Briefly, cells were seeded at a density of 1.2 3 104 cells/well in 96-well culture plates and allowed to adhere for 24 h before incubation in low (0.5% v:v FBS) serumcontaining DMEM for 16 h. Cells were treated for 24 h with sera (1% final concentration) pooled (equal volumes) from PND 50 rats fed either CAS (n 5 10) or WPH (n 5 10). Metabolically active cells, measured by the reduction of MTS tetrazolium compound into formazan (CellTiter 96 AQueous nonproliferation assay kit) were quantified from absorbance readings at 570 nm using a microplate reader. Results were calculated as the average absorbance readings from 2 experiments, with 6 individual wells per treatment assayed per experiment. MCF-7 cells, seeded at a density of 1 3 105 /well and serumstarved overnight, were treated with CAS sera (1% final concentration) in the presence or absence of recombinant rat MCP-1 (BD Biosciences; 10 mg/L), WPH sera (1% final concentration), or recombinant MCP-1 (10 mg/L) in FBS (1%). Cells were processed for TUNEL as described above or harvested for isolation of RNA and gene expression analysis. The primer sequences for the human Tp53 and p21 genes are as follows (Forward and Reverse, respectively): 1) Tp53 (59GGC GCA CAG AGG AAG AGA AT-39; 59-GGA GAG GAG CTG GTG TTG TTG-39); and 2) p21 (59-GAC AGC AGA GGA AGA CCA GTG G-39; 59-GGC GTT TGG AGT GGT AGA AAT C-39). The concentration of recombinant MCP-1 was selected on the basis of the initial dose-response studies using 1, 10, and 100 mg/L; the 10mg/L dose resulted in the greatest inhibition of Tp53 transcript levels and was used in subsequent studies (data not shown). Cytokine antibody array and MCP-1 ELISA. Rat cytokine antibody array membranes and kits were used following the manufacturer’s instructions (RayBiotech). The membranes, which contain biotinconjugated antibodies for 18 cytokines, were incubated with sera pooled from PND 50 rats fed CAS (n 5 10) or WPH (n 5 10), and used at a final concentration of 10%. Signals were detected and quantified using the BioRad molecular analyst detection system (BioRad). MCP-1 levels in serum from PND 50 rats fed CAS (n 5 12) and WPH (n 5 14) from this and a previous study (7) were quantified using a specific rat MCP-1 ELISA kit (BioSource International). Data are reported as means (ng/L) 6 SEM.


cellular biosynthesis of glutathione (GSH), which can boost the immune system and detoxify potential carcinogens (23,24). In an earlier work, we showed that the reduced mammary tumor incidence and increased mammary tumor latency in the lifetime of 7,12-dimethylbenz[a]anthracene (DMBA)-treated female rats exposed to WPH compared with CAS (control)-based diets were associated with increased differentiation and enhanced PTEN gene expression in mammary epithelial cells (7). Given that WPH is a collection of proteins with predictably different biological activities (23), distinct signaling pathways likely collaborate to confer its mammary tumor protective effects. In the present study, we evaluated the hypothesis that mammary tissues from WPH-fed rats are more effectively protected from DNA damage induced by endogenous (metabolic) stressors than those fed the control diet, CAS, due in part to enhanced serum proapoptotic and immune surveillance activity with dietary WPH intake. To address this, we did the following: 1) evaluated the activation (phosphorylation) status of Tp53, a hallmark of DNA damage response, in mammary epithelial cells of young adult rats fed CAS or WPH diets for their entire lifetime; 2) examined the proapoptotic activity of sera from these rats toward mammary tumor MCF-7 and normal (nontumor) MCF-10A cells; and 3) measured the serum levels from rats of the 2 diet groups of the chemokine monocyte chemoattractant protein (MCP)-1 whose expression is associated with increased immune surveillance and antimalignant activity (25,26).

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Statistical analysis. Data from all studies are expressed as means 6 SEM. Differences between diet groups were analyzed for statistical significance by Student’s t test, or one-way ANOVA, followed by Dunn’s test (where each treatment was compared to CAS controls), as indicated in each figure legend. Differences with P # 0.05 were considered significant.

RESULTS

FIGURE 2 Expression levels of total and phosphorylated Tp53 and p38 MAPK proteins in mammary glands of PND 50 rats fed CAS- and WPH-based diets. (A) Western blot analysis of nuclear extracts of mammary tissues; each lane represents an individual rat. (B) Graphical representation of levels of immunoreactive P-Tp53Ser15 and P-p38 MAPK. (C) Graphical representation of levels of immunoreactive Tp53 and p38 MAPK. Values are means 6 SEM, n ¼ 4. *Different from CAS, P , 0.05.

MCP-1 in CAS- and WPH-fed rat sera. Array membranes incubated with WPH rather than with CAS sera had higher signal intensities for MCP-1 (data not shown). To confirm this, a specific ELISA for rat MCP-1 was used to measure serum MCP-1 concentrations in CAS- and WPH-fed PND 50 females. Serum from the WPH group had higher MCP-1 levels (183.52 6 16.4 ng/L) than that from the CAS group (134.44 6 15.64 ng/L) (P 5 0.041) The percentage of apoptotic cells in MCF-7 cells incubated for 24 h in CAS serum (1%) 1 MCP-1 (10 mg/L) was significantly increased (P , 0.001) over that of cells incubated in CAS serum alone, and was comparable to that of cells treated with WPH serum (1%) or MCP-1 (in 1% FBS) (Fig. 4A). The reduction in Tp53 and p21 transcript levels of MCF-7 cells treated with WPH serum relative to cells treated with CAS serum was also mimicked by recombinant MCP-1, added alone (10 mg/L) or with CAS serum (Fig. 4B, C).

DISCUSSION FIGURE 1 Expression of growth-arrest, apoptotic, and DNA damage repair genes in mammary glands of PND 50 rats fed CAS- or WPHbased diets (A, B). Levels of RNA were normalized to 18S rRNA. Values are means 6 SEM, n ¼ 8–10. *Different from CAS, P , 0.05.

We reported previously that the protective effects of lifetime dietary exposure to WPH against mammary tumor development in female rats exposed to DMBA were associated with


Mammary expression of growth arrest, apoptotic, and DNA damage repair genes of CAS- and WPH-fed rats. The levels of Tp53 mRNA did not differ in mammary tissues of rats fed the 2 diets (Fig. 1A). However, p21 (P 5 0.008), wip1 (P 5 0.012), Sesn1 (P 5 0.025), Mgmt (P , 0.001), BRCA1 (P 5 0.065), and Bax (P , 0.001) genes had lower expression levels in mammary glands of rats fed WPH than in those fed CAS (Fig. 1A, B). GADD153/CHOP and PTEN transcript levels were not affected by the diets (Fig. 1B). Although total Tp53 protein levels did not differ between the diet groups, those of P-Tp53Ser15 protein were reduced (P , 0.05) in mammary glands of rats fed WPH relative to CAS-fed rats (Fig. 2). Activated p38 MAPK levels were also lower (P , 0.05) in mammary tissues of WPH- than of CAS-fed rats, in the absence of differences in total p38 MAPK protein levels (Fig. 2). Apoptotic activity of rat sera as a function of diet. MCF-7 cells treated with WPH sera had lower (P 5 0.029) cell viability than those incubated with control CAS sera (Fig. 3A). Further, these cells had a higher percentage of apoptosis (P , 0.001) when grown in the presence of WPH than CAS sera (Fig. 3B, C). By contrast, nontumorigenic MCF-10A cells, treated with either CAS or WPH sera, did not exhibit detectable apoptosis (Fig. 3C). The higher apoptotic status of WPH-treated, relative to CAS-treated MCF-7 cells was accompanied by fewer (P , 0.05) transcripts (mRNA arbitrary units) for Tp53 (CAS, 1.74 6 0.17; WPH, 0.72 6 0.10) and p21 (CAS, 2.13 6 0.43; WPH, 1.21 6 0.25).

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higher mammary differentiation status at the time of carcinogenic insult (7). The present study examined signaling pathways that may underlie the reduced tumor incidence with dietary WPH. Because the functions of the tumor suppressor Tp53 are intimately associated with normal cellular response to DNA damage and stress signals (11,12,14), we posited that evaluation of the expression levels of activated Tp53 and of genes involved in Tp53-mediated apoptosis (Bax, PTEN), growtharrest (p21, wip1), and DNA damage repair (Mgmt, BRCA1, GADD153, Sesn1) may provide insights into the early influence of diet on the stress status of mammary cells before a secondary (carcinogenic) insult and may relate to dietary antitumor effects. Here we report that mammary tissues from young adult female rats fed a lifetime diet of WPH displayed reduced genotoxic stress response parameters relative to those of their CAS-fed counterparts, suggesting increased protection and hence, less susceptibility, of these tissues from endogenous DNA damage with dietary WPH exposure. In particular, we showed that mammary tissues of WPH-fed rats relative to those fed CAS, had significantly lower expression levels of activated Tp53 (P-Tp53Ser15) and of the Tp53 target genes p21, wip1, Bax, Sesn1, BRCA1, and Mgmt, and exhibited attenuated p38 MAPK signaling. Given the protein heterogeneity of WPH (23), it was not possible in this study to identify the molecular component(s) in whey that might be responsible for the observed reduction in mammary p38 MAPK signaling, leading to decreased Tp53 activation. However, the induction of the cellular GSH antioxidant system by one or more of the whey proteins may be involved because the lower phosphorylation status of p38 MAPK, as noted in the present study, has been linked to decreased oxidative stress and higher GSH levels (28). The observed increase in apoptotic status of MCF-7 tumor cells when treated with WPH compared with CAS sera suggests the presence of serum proapoptotic factor(s) whose synthesis is influenced by diet. We found similar proapoptotic activity of serum from rats fed diets containing soy proteins or supplemented with the isoflavone genistein toward MCF-7 cells (10).

FIGURE 4 Effects of recombinant MCP-1 (10 mg/L) on apoptosis and gene expression in serum-treated MCF-7 cells. (A) The percentage of apoptotic cells is expressed as the percentage of immunofluoresecent cells over the total numbers of cells counted. Levels of Tp53 (B) and p21 (C) transcripts were normalized to 18S rRNA. Values are means 6 SEM, n ¼ 4–6. *Different from CAS, P , 0.05.

These results imply that the early elimination of damaged cells, thereby preventing their further proliferation and fixation of DNA damage as mutations, may underlie in part the protective effects of WPH diet against an additional (secondary) carcinogenic insult. The reduction in Tp53 and p21 expression levels of MCF-7 cells coincident with their increased sensitivity to apoptosis suggests that the apoptotic response of tumor cells may be mediated by attenuating Tp53 expression or activity. Although the latter may appear incongruous to the role of Tp53 as a read-out for decreased DNA damage in normal mammary glands, it is worth noting that cellular context (normal vs. tumor cells) including the contribution of the stromal compartment to the response of epithelial cells to apoptotic or tumorigenic stimulus (29) may underlie these differences. Importantly, the cellular Tp53 response of mammary tumor cells to WPH serum agrees with 2 previous reports on dietary factors with purported tumor protective activities. In one study, the induction of apoptosis of several types of cancer cells in vitro by the dietary factor curcumin, which exhibits antioxidant, anti-inflammatory, and anticarcinogenic properties, involved the downregulation of Tp53 expression (30). In a second study, short-chain fatty acids generated from the digestion of fibers from bran and wheat were also shown to downregulate Tp53 mRNA levels during apoptosis, a process mediated by the stress-activated protein


FIGURE 3 Effects of serum from PND 50 rats fed CAS- or WPH diets on MCF-7 cell viability and apoptosis. (A) Cell viability. (B) The proportion of apoptotic cells is expressed as the percentage of immunofluorescent cells over the total numbers of cells counted. (C) The bright field illustrates the comparable numbers of MCF-7 and MCF-10A cells treated with CAS or WPH serum that were analyzed for the presence of fluorescence signals. Values are means 6 SEM, n ¼ 4–6. *Different from CAS, P , 0.05.

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ACKNOWLEDGMENT The authors thank Frank A. Simmen for critical reading of the manuscript and helpful discussions during the course of this study.

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kinase, c-jun N-terminal kinase 1 (31). Thus, the identification of diet-induced systemic factors with the ability to selectively kill tumor cells in part, by downregulating Tp53 expression, may provide novel therapeutic strategies for cancer. We identified the chemokine MCP-1 as a serum factor that may be partly responsible for the increased proapoptotic activity of WPH relative to CAS sera in MCF-7 cells. Our suggestion is based on the higher circulating levels of MCP-1 in WPH-fed than in CAS-fed rats and the ability of recombinant MCP-1 to mimic the effects of WPH serum in increasing apoptosis and in decreasing Tp53 and p21 gene expression in MCF-7 cells when added alone or in combination with CAS serum. Given the immunomodulatory role of MCP-1 via its putative effects on innate and acquired immune responses (25,26,32), our findings indicate that an active immune effector response elicited by dietary WPH may contribute to its protective effects against chemically induced tumorigenesis (7). The possibility of an association among MCP-1, mammary tumor status, and immune surveillance is supported by the reported downregulation of MCP-1 expression in human and mouse mammary tumors (33), the presence of functional MCP-1 receptor in mammary cells and tissues (34), and inhibition by estrogen, a breast cancer risk, of MCP-1 gene expression and bioactivity (34,35). Although the source of serum MCP-1 and the mechanism underlying its effect on gene expression in mammary tumor cells leading to apoptosis are presently unknown, the possibility that MCP-1 may serve as a useful marker for diet-induced immune protection warrants further investigation using animal models and in human subjects consuming whey as part of a normal diet. In summary, we demonstrated, using activated (phosphorylated) Tp53 as a read-out, that mammary tissues from rats fed a WPH diet are more protected from DNA damage than their CAS-fed counterparts. The postulated higher genomic integrity of mammary cells with a WPH diet, as supported by the reduction in activated p38 MAPK signaling, activated Tp53 levels, and expression of DNA damage repair, proapoptotic, and growth-arrest genes, suggests that the higher stressresistant status of mammary tissues before exogenous carcinogenic exposure can influence the outcome of mammary tumor development. We suggest that identification of the dietary WPH component(s) that mediates the desensitization of mammary cells to DNA damage and increases systemic proapoptotic and/or immune surveillance activity has potentially important benefits for the prevention and treatment of mammary and other types of cancer.