Detection of ebaf, a novel human gene of the transforming growth factor beta superfamily association of gene expression with endometrial bleeding.

Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.

Adhesion to fibronectin via beta1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR).

The tumor cell environment may influence drug response through interactions with the extracellular matrix (ECM). We recently reported that adhesion of myeloma cells to fibronectin (FN) via beta1 integrins is associated with a cell adhesion mediated drug resistance (CAM-DR). Activation of beta1 integrins is known to influence both apoptosis and cell growth. We hypothesized that the FN mediated cytoprotection may be in part due to perturbations in cell cycle progression. In this report we demonstrate that adhesion of myeloma cells to FN results in a G1 arrest associated with increased p27kip1 protein levels and inhibition of cyclin A and E associated kinase activity. Disruption of cells from FN adhesion resulted in a rapid recruitment of cells into S phase, a decrease in p27kip1 levels, and reversion to a drug sensitive phenotype. Treatment of cells with p27Kip1 antisense oligonucleotides did not affect FN adhesion; however, p27Kip1 protein levels were reduced and cells became sensitive to cytotoxic drugs. These studies demonstrate that beta1 mediated adhesion of myeloma cells to FN regulates p27kip1 levels and that p27kip1 levels are causally related to CAM-DR. Disruption of beta1 integrin mediated FN adhesion may represent a potential target for the potentiation of drug induced apoptosis.

Clonal variability in CD95 expression is the major determinant in Fas-medicated, but not chemotherapy-medicated apoptosis in the RPMI 8226 multiple myeloma cell line.

CD95 (Fas/APO-1) is a member of the TNFR superfamily that induces apoptosis following cross-linking with its cognate ligand, CD95L (FasL/APO-1L) or agonist antibody. The human myeloma cell line, RPMI 8226, has limited sensitivity to CD95-mediated apoptosis, with a maximum of 65% of the population responding. To determine the source of the limited sensitivity to CD95-mediated apoptosis, we isolated multiple clones from the RPMI-8226 cell line by limiting dilution. Analysis of these clones demonstrated that sensitivity to CD95-mediated cell death directly correlated with CD95 expression. Clones with high levels of CD95 expression had greater than 90% cell death, whereas cells with low levels of expression had less than 10% cell death. In contrast, no correlative differences were identified for other members of the DISC complex, or for members of the anti-apoptotic Bcl-2 family. We further examined the sensitivity of the 8226 clones to various cytotoxic agents. Although modest clonal variability was demonstrated in response to the chemotherapeutic drugs, doxorubicin, etoposide (VP-16), and vincristine, there was no correlation between CD95 function and sensitivity to chemotherapeutic drugs. These results indicate that in this cell line, receptor expression is rate limiting in CD95-mediated apoptosis, whereas CD95 expression was not a determinant in drug-induced programmed cell death.

Distinct tumor specific expression of TGFB4 (ebaf)*, a novel human gene of the TGF-beta superfamily.

We recently identified a novel gene of the TGF-beta superfamily, endometrial bleeding associated factor, TGFB4 (ebaf), that, throughout the menstrual cycle, exhibited a defined expression in human endometrium. Here, we report on the expression of TGFB4 (ebaf) in normal and neoplastic human tissues. The expression of this gene was absent in a host of normal tissues including lung, stomach, small bowel, liver, kidney, breast, lymph node, spleen, ovary and fallopian tube. However, a weak expression of the 2.1 kb variant of the TGFB4 (ebaf) mRNA was observed in rectal, ovarian, and testicular tissues and the 2.1 and 2.5 kb TGFB4 (ebaf) mRNAs were observed in the pancreatic tissue. The expression of the mRNA of this gene was absent in sarcomas, Hodgkin's and non-Hodgkin's lymphomas, melanomas, squamous cell carcinomas, hepatocellular carcinomas, renal cell carcinomas, and adenocarcinomas of the breast, endometrium and lung. The expression of the TGFB4 (ebaf) mRNA was observed primarily in adenocarcinomas that exhibited a mucinous differentiation. This included colonic, duodenal, and ovarian adenocarcinomas. The expression of TGFB4 (ebaf) mRNA was absent in non- mucinous colonic, gastric and ovarian adenocarcinomas and adenocarcinomas of colon metastatic to the liver. However, some serous adenocarcinomas of the ovary also exhibited TGFB4 (ebaf) mRNA. The testicular tumors, seminomas and embryonal carcinomas, also expressed TGFB4 (ebaf) mRNA. These findings show that the TGFB4 (ebaf) mRNA has distinct tumor specific expression.

CD95 antigen mutations in hematopoietic malignancies.

The CD95 receptor, also known as Fas/Apo-1, is a member of the Tumor Necrosis Factor receptor (TNF-R) family of death receptors. Apoptosis mediated by CD95 plays a central role in maintaining homeostasis of the immune system. Dysregulation of the CD95 apoptotic pathway has been proposed as a mechanism of oncogenesis by providing a survival advantage to potentially malignant cells. This extended lifespan could allow the accumulation of further mutations leading to malignant transformation. Several mechanisms of resistance to CD95 mediated apoptosis have been identified, including reduced surface expression of the receptor, overexpression of anti-apoptotic molecules, and loss of function mutations. This review will focus on the potential role of the CD95-CD95 ligand system in the pathogenesis of hematological malignancies, with particular emphasis on recent work from our laboratory examining the expression of CD95 in B cell lymphomas. We demonstrate that CD95 mutations occur at low frequency in NHL tumors, however, surface expression of the CD95 protein varies with the subtype of lymphoma. Loss of surface CD95 is more likely to occur in lymphomas of aggressive histology, and is unrelated to the detection of CD95 mutations.

Mutations in the Fas antigen in patients with multiple myeloma.

Programmed cell death, or apoptosis, is well documented as a physiological means of eliminating activated lymphocytes and maintaining immune homeostasis. Apoptosis has also been implicated in the targeting of tumor cells by cytotoxic T lymphocytes and natural killer cells. One of the two primary mechanisms used in cell-mediated cytotoxicity is the Fas/FasLigand system. Activated or transformed cells expressing the Fas antigen on their surface are susceptible to killing by immune effector cells that express the Fas ligand. Many neoplastic cells, including those derived from patients with multiple myeloma, express Fas antigen on their surface, but do not undergo apoptosis in response to antigen crosslinking. One possibility for the lack of Fas-mediated apoptosis includes mutations in the Fas antigen. Loss of function mutations in the Fas antigen have been associated with congenital autoimmune disease in humans, and have been defined as the genetic defect the in lpr mice. Mutations in the Fas antigen have not been previously described in cancer patients. In this study, we show that mutations occur in the Fas antigen which may cause loss of function and contribute to the pathogenesis of the neoplastic disease, multiple myeloma. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis, and DNA sequencing, we examined the cDNA structure of the Fas antigen in 54 bone marrow (BM) specimens obtained from myeloma patients. Six patient specimens (11%) did not express detectable levels of Fas antigen mRNA. Of the 48 BM specimens which did express Fas antigen, 5 (10%) displayed point mutations. All of the mutations identified were located in the cytoplasmic region of the Fas antigen known to be involved in transduction of an apoptotic signal. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome, ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lprcg mouse. Although the functional status of these point mutations remains to be determined, we propose that Fas antigen mutations may contribute to the pathogenesis and progression of myeloma in some patients.

Myeloma cells selected for resistance to CD95-mediated apoptosis are not cross-resistant to cytotoxic drugs: evidence for independent mechanisms of caspase activation.

We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95-mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and drug-induced apoptosis occurs only when cytotoxic drugs are used as the selecting agent. Using the inverse approach, we transfected the CD95-negative cell line, K562, with a CD95 expression vector. Clones expressing variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95 surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show that CD95 and chemotherapeutic drugs use common apoptotic effectors, but the point of convergence in these two pathways is downstream of CD95 receptor/ligand interaction.