Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells.

We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.

Production of mammastatin, a tissue-specific growth inhibitor, by normal human mammary cells.

The growth of human mammary cells may be regulated by a balance between growth stimulatory and growth inhibitory pathways. Polypeptides of 47 and 65 kilodaltons (mammastatin) were isolated from conditioned medium of normal human mammary cells. Monoclonal antibodies against mammastatin were generated that blocked its activity and were used for purification and further characterization of the protein. Mammastatin inhibited the growth of 5 transformed human mammary cell lines, but had no effect on the growth of 11 transformed human cell lines derived from nonmammary tissues. Mammastatin appeared to be a heat-labile protein distinct from transforming growth factor-beta (TGF-beta). By immunoperoxidase staining it was detected in cultured normal human mammary cells, but was decreased in transformed mammary cells.

Symmetric division of cancer stem cells--a key mechanism in tumor growth that should be targeted in future therapeutic approaches.

As cancer stem cells (SCs) drive tumor growth, it is only through the elimination of those cancer SCs that a pharmacologic cure can be attained. To study ways to develop drugs that target cancer SC, we investigated changes in cellular mechanisms and kinetics that occur in SC populations during colorectal cancer (CRC) development. We used computer modeling to determine which changes could give rise to exponential increases in both SC and non-SC populations in CRC. Our results show that the only mechanism that can explain how these subpopulations increase exponentially in CRC development involves an increase in symmetric SC cell division. This finding suggests that any systemic therapies designed to effectively treat CRC and other cancers must act to control or eliminate symmetrical cancer SC division in tumors, while minimally affecting normal SC division in non-tumor tissues.

Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol. Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0). Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge. By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells. Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2. Cumulative suspension cells on ECM dishes were eight times controls. ECM influenced both hematopoietic progenitor cell proliferation and differentiation. Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05). There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points. This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment.

Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.

EMP2 is a novel therapeutic target for endometrial cancer stem cells.

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against cancer stem cells in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells.

Cell surface alpha-D-galactopyranosyl end groups: use as markers in the isolation of murine tumor cell lines with different cancer-causing potentials.

Cell lines from 2 3-methylcholanthrene-induced murine tumors were established in culture and examined for reactivity with Griffonia simplicifolia isolectin B4 (GS I-B4), a lectin that has strict specificity for terminal alpha-D-galactopyranosyl residues. Virtually all of the cells in both populations were strongly reactive, indicating the presence of this carbohydrate on the cell surface. Both tumor lines were exposed to human serum with antibodies to the blood group B antigen. More than 99.99% of the exposed cells were killed by this treatment. This is not surprising, because terminal alpha-D-galactopyranosyl residues comprise the blood group B antigen. From the few surviving cells, it was possible to establish cell lines resistant to the cytotoxic effects of the anti-blood group B antibodies. A total of 4 cell lines were independently obtained in this way. The human serum-resistant lines showed no detectable reactivity with GS I-B4, indicating that alpha-D-galactopyranosyl-deficient cell lines had been obtained. The parent and variant cells were compared for reactivity with anti-laminin antibodies. Both parent lines showed strong reactivity by immunofluorescence in the viable state, whereas the alpha-D-galactopyranosyl-deficient lines showed no reactivity. The parent and variant lines were also compared with regard to in vitro and in vivo growth. The alpha-D-galactopyranosyl-deficient lines had reduced in vitro growth capacity relative to the parent lines. More importantly, in contrast to the parent lines, these lines were significantly less tumorigenic than the parent lines when injected into syngeneic mice and did not metastasize spontaneously.

Collagen-production inhibitors evaluated as antitumor agents.

Proline analogues such as cis-4-hydroxy-L-proline (CHP) and L-azetidine-2-carboxylic acid (A2C) were tested for their antitumor activity in tissue culture and in vivo. In culture, CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen. CHP appeared to selectively inhibit collagen biosynthesis with only a slight effect on protein synthesis. Culturing cells on type IV collagen matrix did not alter the antiproliferative effect of CHP. The inhibition of 450.1 mouse mammary tumor cells was fully reversible when cultures were incubated for 6 or 12 hours with 25 micrograms CHP/ml but was irreversible after 24 hours of exposure. Of the proline analogues tested against 450.1 tumor cells, A2C and CHP were the most potent inhibitors of cell growth. These two compounds were therefore tested in vivo using 3 transplantable tumors, all of which synthesized basement-membrane collagen. CHP and A2C were given twice daily to mice for 7 to 10 days at doses ranging from 50 mg/kg (body wt) to 600 mg/kg (body wt) per injection. Both CHP and A2C were completely inactive against the 450.1 mammary tumor and the EHS sarcoma. Both compounds also caused considerable liver toxicity. Against CD8F1 mammary tumors, treatment with maximum tolerated doses of CHP and A2C resulted in a slight but insignificant inhibition of tumor growth. While our studies confirmed previous findings that CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen, CHP and A2C did not appear to be efficacious antitumor agents.

Unsaturated fatty acid requirements for growth and survival of a rat mammary tumor cell line.

A cell line, the growth and survival of which is markedly affected by linoleic acid, has been established from a carcinogen-induced rat mammary tumor. The cells have been continuously passaged in 5% rat serum plus 10% fetal calf serum-supplemented medium. The rat serum component was found to be indispensalbe, for when it was omitted the growth rate rapidly declined and the cells died by 5 to 7 days. Removal of the rat serum from the growth medium also resulted in a dramatic loss of Oil Red O-positive droplets in the cells, suggesting that the lipid component of rat serum might be a major growth-promoting principle in rat serum. This is likely since the total lipid fraction, but not the delipidized protein fraction, could largely supplant requirement of the cells for rat serum. Pure linoleic acid was found to be effective in maintaining the cell growth in delipidized serum or in whole fetal calf serum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in rat serum than in fetal calf serum.

bcl-xs gene therapy induces apoptosis of human mammary tumors in nude mice.

Bcl-xs is a dominant negative repressor of Bcl-2 and Bcl-xL, both of which inhibit apoptosis. We used a replication-deficient adenoviral vector to transiently overexpress Bcl-xs in MCF-7 human breast cancer cells, which overexpress Bcl-xL. Infection with this vector induced apoptosis in vitro. We then determined the effects of intratumoral injection of bcl-xs adenovirus on solid MCF-7 tumors in nude mice. Tumors injected four times with the bcl-xs adenovirus showed a 50% reduction in size. Using terminal transferase-mediated dUTP-digoxigenin nick end labeling, we observed apoptotic cells at sites of bcl-xs adenoviral injection. These experiments demonstrate the feasibility of using bcl-xs gene therapy to induce apoptosis in human breast tumors.

Sensitivity of N-nitrosomethylurea-induced rat mammary tumors to cis-hydroxyproline, an inhibitor of collagen production.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Overexpression of Bcl-XS sensitizes MCF-7 cells to chemotherapy-induced apoptosis.

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy. We report that Bcl-XL, which functions like Bcl-2 to inhibit apoptosis, is highly expressed in MCF-7 human breast carcinoma cells. We used Bcl-XS, a dominant negative inhibitor of Bcl-2 and Bcl-XL, to demonstrate the role of these genes in modulating chemotherapy-induced apoptosis. Bcl-XS overexpressed in MCF-7 cells by stable transfection does not affect viability by itself but induces a marked increase in chemosensitivity to VP-16 or taxol. Using an ELISA assay which quantitates DNA damage, we demonstrate that this sensitization is due to apoptosis, suggesting the therapeutic utility of targeting this pathway.

Breast conservation and prolonged chemotherapy for locally advanced breast cancer: the University of Michigan experience.

PURPOSE: To determine whether breast conservation and prolonged neoadjuvant chemotherapy have efficacy in locally advanced breast cancer (LABC), as measured by survival and rate of breast conservation. MATERIALS AND METHODS: Eighty-nine patients with stage III disease were enrolled at the University of Michigan (UM) onto a prospective nonrandomized trial. Patients received nine 21-day cycles of neoadjuvant chemohormonal therapy that consisted of doxorubicin 30 mg/m2 and cyclophosphamide 750 mg/m2 intravenously on day 1, conjugated estrogens 0.625 mg orally twice daily on days 6 to 8, methotrexate 40 mg/m2 and fluorouracil 500 mg/m2 intravenously on day 8, and tamoxifen 10 mg orally twice daily on days 9 to 14. Patients with a negative biopsy received radiation only, while those with residual disease underwent mastectomy and postoperative radiotherapy. Eight more cycles of chemohormonal therapy were administered after local-regional therapy. RESULTS: The clinical response rate to neoadjuvant therapy was 97%, 28% of patients had a complete pathologic response evaluated at biopsy. Five-year overall and disease-free survival probabilities were 54% and 44%, respectively. The median disease-free survival time was 2.4 years. The 5-year actuarial rates of local-regional control with local failure as only first failure were 82% and 78% following radiotherapy, and mastectomy and radiotherapy, respectively (P = .99). CONCLUSION: Prolonged neoadjuvant chemohormonal therapy and biopsy-driven local therapy have efficacy in LABC, with 28% of patients being candidates for breast conservation and a 5-year overall survival rate of 54%.

Evaluating the financial impact of clinical trials in oncology: results from a pilot study from the Association of American Cancer Institutes/Northwestern University clinical trials costs and charges project.

PURPOSE: Medical care for clinical trials is often not reimbursed by insurers, primarily because of concern that medical care as part of clinical trials is expensive and not part of standard medical practice. In June 2000, President Clinton ordered Medicare to reimburse for medical care expenses incurred as part of cancer clinical trials, although many private insurers are concerned about the expense of this effort. To inform this policy debate, the costs and charges of care for patients on clinical trials are being evaluated. In this Association of American Cancer Institutes (AACI) Clinical Trials Costs and Charges pilot study, we describe the results and operational considerations of one of the first completed multisite economic analyses of clinical trials. METHODS: Our pilot effort included assessment of total direct medical charges for 6 months of care for 35 case patients who received care on phase II clinical trials and for 35 matched controls (based on age, sex, disease, stage, and treatment period) at five AACI member cancer centers. Charge data were obtained for hospital and ancillary services from automated claims files at individual study institutions. The analyses were based on the perspective of a third-party payer. RESULTS: The mean age of the phase II clinical trial patients was 58.3 years versus 57.3 years for control patients. The study population included persons with cancer of the breast (n = 24), lung (n = 18), colon (n = 16), prostate (n = 4), and lymphoma (n = 8). The ratio of male-to-female patients was 3:4, with greater than 75% of patients having stage III to IV disease. Total mean charges for treatment from the time of study enrollment through 6 months were similar: $57,542 for clinical trial patients and $63,721 for control patients (1998 US$; P =.4) CONCLUSION: Multisite economic analyses of oncology clinical trials are in progress. Strategies that are not likely to overburden data managers and clinicians are possible to devise. However, these studies require careful planning and coordination among cancer center directors, finance department personnel, economists, and health services researchers.

Treatment of metastatic cancer with tetrathiomolybdate, an anticopper, antiangiogenic agent: Phase I study.

Preclinical and in vitro studies have determined that copper is an important cofactor for angiogenesis. Tetrathiomolybdate (TM) was developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. Given the potency and uniqueness of the anticopper action of TM and its lack of toxicity, we hypothesized that TM would be a suitable agent to achieve and maintain mild copper deficiency to impair neovascularization in metastatic solid tumors. Following preclinical work that showed efficacy for this anticopper approach in mouse tumor models, we carried out a Phase I clinical trial in 18 patients with metastatic cancer who were enrolled at three dose levels of oral TM (90, 105, and 120 mg/day) administered in six divided doses with and in-between meals. Serum ceruloplasmin (Cp) was used as a surrogate marker for total body copper. Because anemia is the first clinical sign of copper deficiency, the goal of the study was to reduce Cp to 20% of baseline value without reducing hematocrit below 80% of baseline. Cp is a reliable and sensitive measure of copper status, and TM was nontoxic when Cp was reduced to 15-20% of baseline. The level III dose of TM (120 mg/ day) was effective in reaching the target Cp without added toxicity. TM-induced mild copper deficiency achieved stable disease in five of six patients who were copper deficient at the target range for at least 90 days.

SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model.

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

A method of limited replication for the efficient in vivo delivery of adenovirus to cancer cells.

Replication-deficient viral vectors are currently being used in gene transfer strategies to treat cancer cells. Unfortunately, viruses are limited in their ability to diffuse through tissue. This makes it virtually impossible to infect the majority of tumor cells in vivo and results in inadequate gene transfer. This problem can be addressed by allowing limited viral replication. Limited viral replication facilitates greater penetration of virions into tissue and can improve gene transfer. We have developed a strategy of limited viral replication using AdRSVlaclys, a chemically modified E1-deleted adenovirus, to codeliver an exogenous plasmid encoding the adenovirus E1 region. This system allows one round of viral replication. We examined the effect of this limited adenovirus replication in vitro and in vivo. In culture, codelivery of virus and pE1 resulted in a large increase in infected cells when compared with control cells exposed to virus and pUC19. In experiments on nude mice bearing HeLa ascites tumors, intraperitoneal injection of AdRSVlaclys/pE1 resulted in a significantly higher percentage of infected HeLa cells as compared with the PBS controls (p < 0.05) or the AdRSVlaclys/pUC19 controls (p < 0.01). These data demonstrate that the transcomplementation of replication-deficient adenovirus with exogenous E1 DNA leads to limited replication, and this controlled replication enhances gene transfer efficiency of adenovirus in vivo.

A novel, conditionally replicative adenovirus for the treatment of breast cancer that allows controlled replication of E1a-deleted adenoviral vectors.

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER(+) human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER(-) cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1a-deleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.

Clinical protocol. Purging of autologous stem cell sources with bcl-x(s) adenovirus for women undergoing high-dose chemotherapy for stage IV breast carcinoma.

High-dose chemotherapy (HDCT) and autologous bone marrow transplantation (BMT) is frequently used to treat patients with metastatic cancer including breast cancer and neuroblastoma. However, the bone marrow of such patients is often contaminated with tumor cells. Recently, we have found that a recombinant adenovirus vector that contains a bcl-x, minigene (a dominant negative inhibitor of the bcl-2 family), called the bcl-x(s) adenovirus, is lethal to cancer cells derived from epithelial tissues, but not to normal human hematopoietic cells. To determine the mechanism, by which this virus spares normal hematopoietic cells, we isolated normal mouse hematopoietic stem cells and infected them with an adenovirus that contains a beta-galactosidase minigene. Such cells do not express beta-galactosidase, indicating that hematopoietic stem cells do not express transgene encoded by adenovirus vectors based upon the RSV-AD5 vector system. When breast cancer cells mixed with hematopoietic cells were infected with the bcl-x(s) adenovirus, cancer cells were selectively killed by the suicide adenoviruses. Hematopoietic cells exposed to the suicide vectors were able to reconstitute the bone marrow of mice exposed to lethal doses of y-irradiation. These studies suggest that adenovirus suicide vectors may provide a simple and effective method to selectively eliminate cancer cells derived from epithelial tissue that contaminate bone marrow to be used for autologous BMT. We therefore propose to initiate a phase I clinical trial to test the safety of this virus in women with breast cancer undergoing high does chemotherapy and autologous BMT.

A bcl-xS adenovirus selectively induces apoptosis in transformed cells compared to normal mammary cells.

Oncogenes which drive the cell cycle, such as c-myc, can sensitize cells to apoptosis. This suggests the possibility that the expression of genes such as bcl-2 or bcl-xL is required to inhibit apoptosis induced by oncogene expression. We hypothesized that inhibition of Bcl-2/Bcl-xL by the pro-apoptotic Bcl-xS protein, would result in selective induction of apoptosis in mammary carcinoma cells compared to their nontransformed counterparts. Therefore, we compared the effects of Bcl-xS expression delivered by a bcl-xS adenovirus (bcl-xS-Adv) vector, on viability and apoptosis of nontransformed versus transformed mammary epithelial cells. We report that c-myc-transformed murine mammary cells are extremely sensitive to apoptosis induced by the bcl-xS adenovirus (bcl-xS-Adv) vector, whereas immortalized, nontransformed murine mammary cells are relatively resistant to apoptosis induced by this vector. Likewise, human mammary epithelial cells transduced with c-erbB-2 were more sensitive to apoptosis induced by the bcl-xS vector than the nontransformed parental cells. Similar results were obtained when we tested the effects of bcl-xS adenoviral infection on primary normal human mammary epithelial cells and SUM-190 PT cells, (a c-erbB-2 over-expressing human mammary carcinoma cell line) grown on Matrigel. These data are consistent with the hypothesis that inhibition of Bcl-2/Bcl-xL can result in selective killing of cancer cells compared to their nontransformed counterparts.