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.

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.

Identification and functional analysis of 9p24 amplified genes in human breast cancer.

Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16(INK4a), p21(Waf1/Cip1) and p27(Kip1). Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.

HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion.

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors, which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis, tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes, increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive, but not resistant, cell lines, an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis, tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore, the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors.

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.

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.

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.

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.

A bcl-x(S) adenovirus demonstrates therapeutic efficacy in an ascites model of human breast cancer.

OBJECTIVES: To determine whether a Bcl-x(S) adenoviral vector has therapeutic potential in an ascites model of human breast cancer in nude mice. METHODS: Advanced ascites were developed by injecting mice intraperitoneally (IP) with MDA-MB-231 human breast carcinoma cells. Mice received sequential IP injections of the Bcl-x(S) virus or a control lac-Z adenovirus. A third group of mice received no virus. Tumor burden and survival were monitored. Histopathology and necropsies were performed on mice. RESULTS: A single injection of the Bcl-x(S) adenovirus produced no systemic or local toxicity and no abnormal histopathology in normal mice. However, abdominal organs within these mice were transduced with the Bcl-x(S) vector. Adenoviral gene transduction efficiency in MDA-MB-231 ascites was 36+/-6.40% (n = 3). Percent weight change differences revealed that ascites bearing mice injected three times with the Bcl-x(S) vector showed a statistically significant decrease in tumor burden compared with lac-Z-injected mice (n = 7; P = .012 on days 10-15 after the first injection). Mice injected with the Bcl-x(S) vector had significantly greater survival relative to lac-Z-injected mice (n = 7; P = .0004). Bcl-x(S) protein expression was detected in aspirates of mice injected with the Bcl-x(S) vector but not the lac-Z vector. Necropsies revealed that ascites bearing mice injected with Bcl-x(S) vector lacked carcinoma in the peritoneal cavity compared with control mice. CONCLUSION: The Bcl-x(S) adenovirus can reduce tumor burden and increase survival in an ascites model of advanced stage breast cancer.

Differential regulation of apoptosis in normal versus transformed mammary epithelium by lutein and retinoic acid.

We examined the effects of all-trans retinoic acid (ATRA) and lutein (a nonprovitamin A carotenoid), on apoptosis and chemosensitivity in primary normal human mammary epithelial cells, SV40 transformed mammary cells, and MCF-7 human mammary carcinoma cells. ATRA and lutein selectively induced apoptosis in transformed but not normal human mammary cells. In addition, both compounds protected normal cells, but not transformed cells, from apoptosis induced by the chemotherapy agents etoposide and cisplatin. Furthermore, lutein and ATRA selectively increased the ratio of Bcl-xL:Bax protein expression in normal cells but not transformed mammary cells, suggesting a possible mechanism for selective modulation of apoptosis. The differential effects of lutein and ATRA on apoptotic pathways in normal versus transformed mammary epithelial cells may have important implications for chemoprevention and therapy.

Overexpression of Bcl-x(L) promotes chemotherapy resistance of mammary tumors in a syngeneic mouse model.

Bcl-x(L), a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-x(L) to provide chemotherapy resistance to tumors, we transfected the mouse bcl-x(L) gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-x(L) conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-x(L) or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-x(L) did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-x(L)-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-x(L) overexpression can promote resistance to chemotherapy in tumors in vivo.

Inhibition of protein synthesis by didemnin B is not sufficient to induce apoptosis in human mammary carcinoma (MCF7) cells.

Didemnin B (DB) is one member of a class of natural cyclic depsipeptides that display potent cytotoxicity in vitro. The detailed mechanism of action of DB is unknown, although it appears to involve the inhibition of protein biosynthesis. Additional activities of DB have established DB as a rapid and potent inducer of apoptosis in HL-60 cells. Our aim was to determine if the induction of apoptosis by DB is mediated through inhibition of protein synthesis in MCF-7 human breast carcinoma cells. Apoptosis was observed only at > or = 100 nM DB, even though inhibition of protein synthesis occurred at much lower DB concentrations (IC50 = 12 nM). DB-induced apoptosis was mediated by caspase activation, since cleavage of the caspase substrate poly(ADPribose) polymerase was observed as early as 6 hr after DB exposure. Two additional protein synthesis inhibitors, cycloheximide (CHX) and emetine (ET), failed to induce apoptosis at concentrations that completely inhibited protein synthesis. Moreover, DB-induced apoptosis was enhanced only slightly by pre- and co-treatment with CHX and ET. Thus, inhibition of protein synthesis alone was not sufficient to induce apoptosis in these cells. As a measure of antiproliferative potential, DB (1-5 nM) inhibited the colony forming ability of MCF7 cells regardless of pretreatment with CHX. In conclusion, additional effects of DB, independent of protein synthesis inhibition, are proposed to account for its ability to induce apoptosis and prevent cell proliferation.

Diet and risk for breast cancer recurrence and survival.

Dietary factors may influence the risk for breast cancer and also the prognosis following diagnosis and treatment. The aim of this study was to assess whether self-reported prediagnosis diet or other patient factors associated with breast cancer incidence were predictive of recurrence and survival. Patients (n = 149) diagnosed with primary breast cancer between 1989 and 1991 were followed for five or more years. Total energy (hazard ratio (HR) = 1.58, 95%, confidence interval (CI) = 1.05, 2.38) as well as total (HR = 1.46, 95% CI = 1.05, 2.01), saturated (HR = 1.79, 95% CI = 1.05, 3.04), and monounsaturated (HR = 1.65, 95% CI = 1.09, 2.49) fat intakes were associated with increased risk, and energy-adjusted bread and cereal consumption (HR = 0.55, 95% CI = 0.33, 0.93) with decreased risk of recurrence. Both total energy (HR = 1.58, 95% CI = 1.03, 2.43) and polyunsaturated fat (HR = 1.84, 95% CI = 1.09, 3.13) intakes were associated with an increased risk of death. All associations between dietary fat and recurrence and survival attenuated following energy adjustment. Oral contraceptive use (HR = 1.28, 95% CI = 1.03, 1.60), lymph node positive status (HR = 2.36, 95% CI = 1.01, 5.49), and tumor stage (HR = 2.22, 95% CI = 1.02, 4.81) were associated with increased risk of recurrence. Tumor stage (HR = 4.96, 95% CI = 1.86, 13.23), lymph node positive status (HR = 3.31, 95% CI = 1.38, 7.95), and estrogen receptor negative status (HR = 2.46, 95% CI = 1.02, 5.94) were associated with increased risk, and arm muscle circumference (HR = 0.27, 95% CI = 0.09, 0.86) and mammographic utilization (HR = 0.77, 95% CI = 0.61, 0.98) with decreased risk of death. Higher levels of energy, fat intakes, and selected patient characteristics (particularly disease stage and anthropometric indicators of adiposity) appear to increase risk of recurrence and/or shortened survival following the diagnosis of breast cancer.

Immunolymphoscintigraphy in breast cancer: evaluation using 131I-labeled monoclonal antibody B72.3.

Noninvasive axillary lymph node staging was investigated using [131I]murine monoclonal antibody B72.3 in 16 patients with breast cancer scheduled for axillary dissection. [131I]B72.3 was injected into ipsilateral finger webs or around the breast biopsy. Scintigraphy to 72 h and gamma-counting/immunohistochemistry of nodes were performed. Specific antibody uptake (%ID/g) and the ratio of specific:nonspecific antibody uptake were not significantly different in tumor-positive versus tumor-negative nodes, suggesting that [131I]B72.3 is unsuitable to discriminate axillary node tumor involvement.

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.

Targeting cancer cell death with a bcl-XS adenovirus.

Transformation is a complex cellular process that requires several genetic abnormalities. In many cases, one of these abnormalities is an inhibition of PCD, which provides a selective advantage for tumor cells. This has been recently shown in an in vivo model, where overexpression of Bcl-XL, is a crucial step in the progression from hyperplasia to neoplasia and is accompanied by a significant decrease in tumor apoptosis [56]. Frequently, overexpression of a member of the Bcl-2 family results in a block in cell death and appears to nullify many built-in cellular defense mechanisms against cancer. Such a block presents a problem because radiation and chemotherapy, standard cancer treatments, ultimately exert their effect by induction of apoptosis and would also be made less effective. Therefore, to better treat cancer it may be necessary to develop novel methods to overcome the effects of the Bcl-2 family. One way to approach this problem is to target the cause--the molecular machinery that allows a cancer cell to survive. Advances in our understanding of apoptosis has identified the Bcl-2 family as a mediator of most apoptosis pathways, including those initiated by oncogenes, tumor suppressor genes, growth factor withdrawal, and external damaging signals. Therefore, functional inhibition of Bcl-2 family members is lethal to many cancer cells. Using gene transfer technology, we can now deliver genes that accomplish this goal. Further investigation will reveal whether this translates to improved therapy in the future.

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.

Murine granulocytic cell adhesion to bone marrow hemonectin is mediated by mannose and galactose.

Hemonectin (HN) is a bone marrow (BM) protein that promotes specific attachment of immature granulocytes and their precursors within the BM. We report that HN is a glycoprotein containing both mannose and galactose residues, and provide evidence that these carbohydrates mediate granulocytic cell adhesion to HN. Carbohydrate structure was determined by digoxigenin-conjugated lectin binding to HN and indicated the presence of mannose, galactose, sialic acid, and the absence of fucose-linked oligosaccharides. The role of carbohydrates in mediating cell adhesion was examined by chemical and enzymatic deglycosylation. Deglycosylation of HN with trifluoromethanesulfonic acid, which cleaves N- and O-linked oligosaccharides, inhibits 66% of cell attachment to HN, and results in an apparent decrease in molecular weight from 60 to 50 kD. Enzymatic deglycosylation with endo-B-N-acetylglucosaminidase H, which hydrolyzes specific N-linked mannose residues, inhibits 30% of cell adhesion to HN. Finally, the role of these specific sugars in hemonectin-mediated cell adhesion was confirmed with neoglycoprotein blocking. Preincubation of BM cells with mannosyl- and galactosyl-BSA probes produces a dose-dependent inhibition of cell attachment to HN, whereas fucosyl-BSA does not inhibit cell adhesion to HN. These results show that mannose and galactose partially mediate adhesion of BM granulocytes to HN.

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.

cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression.

Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma. Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000). Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library. Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs. No significant sequence similarity was observed on analysis of current GenBank databases. The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody. Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein. This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin. Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function. Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney. On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons. Its tissue-specific expression may suggest a functional role in hematopoiesis.