CAOV3 Ovarian Cancer Cell Research Articles

Tumor promoting properties of the ETS protein MEF in ovarian cancer

We have previously shown that MEF (myeloid ELF1-like factor, also known as ELF4) functions as a transcriptional activator of the interleukin (IL)-8, perforin, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-3 genes in hematopoietic cells. MEF is also expressed in non-hematopoietic tissues including certain ovarian cancer cells. To define the function of MEF in these cells, we examined primary human ovarian epithelial tumors and found that MEF is expressed in a significant proportion of ovarian carcinomas, and in the CAOV3 and SKOV3 ovarian cancer cell lines, but not in normal ovarian surface epithelium. Manipulating MEF levels in these cell lines altered their behavior; reducing MEF levels, using short hairpin RNA expressing vectors, significantly inhibited the proliferation of SKOV3 and CAOV3 cells in culture, and impaired the anchorage-independent growth of CAOV3 cells. Overexpression of MEF in SKOV3 cells (via retroviral transduction) significantly increased their growth rate, enhanced colony formation in soft agar and promoted tumor formation in nude mice. The oncogenic activity of MEF was further shown by the ability of MEF to transform NIH3T3 cells, and induce their tumor formation in nude mice. MEF is an important regulator of the tumorigenic properties of ovarian cancer cells and could be used a therapeutic target in ovarian cancer.

Anti-apoptotic hPEBP4 silencing promotes TRAIL-induced apoptosis of human ovarian cancer cells by activating ERK and JNK pathways

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cell lines but not typically in normal or nontransformed cells, which makes TRAIL a desirable therapeutic agent to fight cancer. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a recently identified anti-apoptotic molecule and has been shown to be highly expressed in breast and ovarian cancer cells. We demonstrate that silencing of hPEBP4 in CaoV-3 ovarian cancer cells potentiates TRAIL-induced apoptosis. We found that endogenous hPEBP4 interacts with Raf-1 and MEK1 in TRAIL-treated CaoV-3 cells by co-immunoprecipitation analysis. Simultaneously, silencing of hPEBP4 in CaoV-3 cells enhances TRAIL-induced ERK and JNK activation. Moreover, the inhibitors of MEK1 or JNK can reduce hPEBP4-silence-induced TRAIL sensitivity. Therefore, silencing of hPEBP4 in CaoV-3 ovarian cancer promotes TRAIL-induced apoptosis, and the increased MAPK activation is required for the apoptosis sensitization. All these data indicate that silencing of hPEBP4, an important potential target, may be a promising approach for the treatment of ovarian cancer.

Maspin Expression Is Characteristic for Cisplatin-Sensitive Ovarian Cancer Cells and for Ovarian Cancer Cases of Longer Survival Rates

High cytoplasmic expression of maspin was described in ovarian cancers of shorter survival rates. Until now, no relationship has been described between expression of maspin and sensitivity to cisplatin in ovarian cancers. This study aimed at examining the relationship between expression of maspin, detected by immunohistochemistry and clinical response to cisplatin in ovarian cancer cases as well as the in vitro sensitivity to cisplatin of 11 ovarian cancer cell lines. The analyzes were performed on 73 samples of ovarian cancer and on A2780P, A2780RCIS, CAOV-3, EFO 21, EFO 27, ES-2, Mdah 2774, OAW 42, OVCAR-3, PA-1, and SKOV-3 ovarian cancer cells. Cytoplasmic maspin expression in studied cells significantly correlated with cisplatin sensitivity. A significantly shorter overall survival and progression-free survival was associated with lower cytoplasmic maspin expression at first-look laparotomies and nuclear maspin expression and secondary cytoreductions. Higher nuclear maspin at first-look laparotomies expression was specific for cases of complete response. In the study, the elevated expression of maspin was shown to be typical for cisplatin-sensitive ovarian cancers.

Th-P15:240 The effect of ageing and obesity on the expression of dyslipidemia in children from FCH families

Statin therapy has been associated with prolonged survival in patients with ovarian cancer. We hypothesized that statins have a cytotoxic effect and that the combination of fluvastatin and cisplatin inhibits cellular proliferation in epithelial ovarian cancer cells.Fluvastatin and cisplatin were examined in CAOV3 and SKOV3 human ovarian cancer cell lines. Cellular proliferation was assessed using MTT assays. Annexin V/propidium iodide (PI) staining was used to discriminate between early and late apoptosis, bromodeoxyuridine and PI staining for cell cycle profiling, and Western blotting for protein expression analysis. Synergy was determined using isobologram analysis.Treatment with combination fluvastatin and cisplatin at multiple doses resulted in significantly greater inhibition of proliferation compared to either drug alone. When examining equipotent combinations of fluvastatin and cisplatin to determine potential synergy, a combination index (CI) of 0.66 was identified for CAOV3 cells and a CI of 0.24 for SKOV3 cells indicating synergy. Combination fluvastatin and cisplatin resulted in G2/M arrest, and a significant increase in early apoptotic cells compared to fluvastatin or cisplatin alone. Moreover, supplementation of farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) demonstrated that GGPP rather than FPP was able to overcome fluvastatin-induced cytotoxicity. Finally, the two-drug combination impaired the expression and modification status of proteins of the Ras pathway.These data demonstrate the synergistic cytotoxicity of fluvastatin and cisplatin, through premature apoptosis and cell cycle arrest, with concomitant dysregulation of Ras pathway proteins. Our studies support a plausible therapeutic role for statins in the adjuvant treatment of ovarian cancer.

Iron chelators deferoxamine and diethylenetriamine pentaacetic acid induce apoptosis in ovarian carcinoma

Objectives. Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Deprivation of iron (Fe), an essential micro-nutrient, by chelation is known to inhibit proliferation of several human cancers but its potential in ovarian cancer treatment remains unknown. We have evaluated the anti-proliferative activities of iron chelators, deferoxamine (DFO), and diethylenetriamine pentaacetic acid (DTPA), in human and rat ovarian cancer cells. Methods. The effect of DFO and DTPA on CaOV-3 (human) and NUTU-19 (rat) ovarian cancer cells was determined by cell proliferation and apoptosis assays (Hoechst staining, DNA fragmentation, and caspase activation), cell cycle analysis, and Fe supplementation studies. Results. DFO and DTPA were cytotoxic to ovarian cancer cells in a dose- and time-dependent manner. DFO inhibited proliferation of NUTU-19 and CaOV-3 cells (IC 50 at 45 and 280 μM, respectively), while DTPA inhibited proliferation of only NUTU-19 cells (IC 50 at 50 μM), at 48 h. DNA synthesis was inhibited in CaOV-3 cells by DFO (>90% at 200 μM) and in NUTU-19 by both DFO and DTPA (>90% at 50 μM). Fe supplementation effectively reversed the cytotoxic effects of DFO and DTPA. Cell cycle analysis showed a G0/G1- and S-phase block with increased apoptosis. DNA fragmentation analysis confirmed apoptosis. Increase in caspase-3, -8, and -9 activities (∼2.4-fold) was associated with apoptosis. Conclusions. Our studies show that Fe chelators suppress ovarian cancer growth by inhibiting proliferation and inducing apoptosis. Therefore, Fe chelators can be potentially developed as novel therapeutic agents to treat ovarian cancer.

PRb2/p130 Decreases Sensitivity to Apoptosis Induced by Camptothecin and Doxorubicin but not by Taxol

In addition to their original function as cell cycle regulators, retinoblastoma (Rb) family members were recently reported to modulate the sensitivity of cancer cells to chemotherapeutic agents. The purpose of this study is to investigate the possible role of pRb2/p130 in the sensitivity of ovarian cancer to camptothecin, doxorubicin, and taxol. pRb2/p130 was overexpressed in the CAOV-3 ovarian cancer cell line, and the effect of pRb2/p130 overexpression on sensitivity to apoptosis trigged by IC(50) doses of different drugs was evaluated by various methods, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Western blot analyses. The results reported in this study support the conclusion that overexpression of pRb2/p130 in the CAOV-3 ovarian cancer cell line lacking wild-type p53 is able to inhibit apoptosis triggered by camptothecin and doxorubicin through the c-Jun NH(2)-terminal kinase signaling transduction pathway. Conversely, taxol-induced cell death is not influenced by the pRb2/p130 protein level. A careful analysis of pRb2/p130 expression in tumor specimens could help to identify the best clinical protocol to be used for each patient, improving efficacy and tolerance and therefore offering additional progress in the treatment of advanced ovarian cancer.

The Kallikrein Gene 5 Splice Variant 2 Is a New Biomarker for Breast and Ovarian Cancer

The presence of more than one mRNA form for the same gene is common among kallikreins, and many of the kallikrein splice variants may hold significant clinical value. The human kallikrein gene 5 (KLK5) is a member of the human kallikrein gene family of serine proteases on chromosome 19q13.4. KLK5 has been shown to be differentially expressed in a variety of endocrine tumors including ovarian, breast and prostate cancer. Utilizing Expressed Sequence Tag database analysis and reverse transcriptase polymerase chain reaction, we identified a new alternatively spliced form of KLK5(KLK5-splice variant 2, KLK5-SV2). This variant mRNA is 1,438 bp in length; formed of 195 bp of 5′ untranslated region, 882 bp of protein coding sequence and a 3′ untranslated region of 326 nucleotides. KLK5-SV2 has 7 exons, the first 2 of which are untranslated, and 6 intervening introns. KLK5-SV2 is different from the classic form of the KLK5 mRNA in its 5′ untranslated region, where the first 5′ untranslated exon of the classic form is split into 2 exons with an intervening intron of 135 nucleotides. KLK5-SV2 is expressed in a variety of tissues, with higher expression levels in the mammary gland, cervix, salivary gland and trachea. The steroid hormone receptor-positive breast cancer cell line BT-474 was used to examine the effect of different steroids on the expression levels of KLK5-SV2. Expression levels were significantly higher after stimulation with androgens, but not estrogens, progestins, aldosterone or corticosteroids. While relatively high levels of expression were found in all 10 normal breast tissues examined, no expression was detected in 16 breast cancer tissues, and expression was significantly lower than normal in the remaining 4 cancers. Expression levels comparable to normal were found in only 1 breast cancer cell line. Weak to no expression was detected in 3 other breast cancer cell lines. KLK5-SV2 was not detectable in any of the 10 normal ovarian tissues examined. It was, however, expressed at relatively high levels in 10 out of 20 ovarian cancer tissues, and lower levels were found in 4 other cancers. No expression was detected in the remaining 6 cancers. High expression levels were also detected in the CAOV-3 ovarian cancer cell line. KLK5-SV2 is a potential biomarker for breast and ovarian cancers.

Evidence for tight coupling of Gi protein–mediated lysophosphatidic acid receptor to stimulated cytokine production in ovarian cancer cell

Objective Lysophosphatidic acid stimulates the proliferation of ovarian cancer cell through specific members of the guanosine triphosphate–binding protein–coupled receptor family. We attempted to identify the guanosine triphosphate–binding protein subtypes that are linked to lysophosphatidic acid receptor-stimulated production of cytokine, which are involved potentially in ovarian cancer development. Study design Cytokine assay kits were used to determine interleukin-6, interleukin-8, and tumor necrotic factor–α that were produced from Caov-3 and SK-OV3 ovarian cancer cell lines. Theα-subunit of Gi was detected by pertussis toxin–catalyzed adenosine diphosphate ribosylation from nicotinamide adenine dinucleotide in isolated plasma membrane. Results Pertussis toxin, but not cholera toxin, brought about adenosine diphosphate ribosylation of Gαi of 41 kd in the plasma membrane. Incubation with lysophosphatidic acid and nonhydrolyzable guanosine triphosphate analog decreased the adenosine diphosphate–ribosylation activity in a dose-dependent manner; a one half–maximal effect occurred with 10 μmol/L lysophosphatidic acid. The apparent inhibition by lysophosphatidic acid of the adenosine diphosphate ribosylation demonstrated that lysophosphatidic acid resolved the α-subunit of the Gi to guanosine triphosphate–bound form in the membranes. Pretreatment of the ovarian cancer cells with the pertussis toxin completely inhibited lysophosphatidic acid–stimulated production of interleukin-6, interleukin-8 and tumor necrosis factor–α. The lysophosphatidic acid–stimulated cytokine production was dose-dependent with a one half-maximal effect at 10 μmol/L. Phosphatidic acid and ceramide 1-phosphate had no effect on the lysophosphatidic acid action on cytokine expression. Conclusion These data demonstrate the coupling of lysophosphatidic acid receptor to Gi protein subfamily in ovarian cancer cell. The Gi that couples lysophosphatidic acid receptor to the effector may define the differences in the signaling pathways of lysophosphatidic acid–activated cytokine expression and proliferation.

Overexpression of Her-2/NEU in epithelial ovarian carcinoma induces vascular endothelial growth factor C by activating NF-kappa B: implications for malignant ascites formation and tumor lymphangiogenesis.

Vascular endothelial growth factor C (VEGF-C) is an important growth factor that governs lymphatic spread and the development of intraperitoneal tumors associated with epithelial ovarian cancer; however, its regulation is not yet understood. Overexpression of Her-2/NEU is related to poor survival in advanced epithelial ovarian carcinoma patients. Accordingly, this study attempted to analyze the association between the Her-2/NEU oncogene and VEGF-C in ovarian carcinoma and to elucidate the molecular mechanism of VEGF-C induction by Her-2/NEU. Immunohistochemistry was used to determine the expression of Her-2/NEU and VEGF-C in tissues from 41 patients with epithelial ovarian carcinoma. Several Her-2/NEU-stably-transfected Caov-3 ovarian carcinoma cells were used to evaluate the effect of Her-2/NEU on VEGF-C, the possible regulation mechanism, and the biological function of VEGF-C. Our experimental results identified a significant association between the Her-2/NEU oncogene and VEGF-C expression in both epithelial ovarian cancer patients (p < 0.05; Fisher's exact test) and in vitro cell lines. The overexpression of Her-2/NEU in Caov-3 ovarian cancer cells resulted in induction of a considerable amount of VEGF-C mRNA and protein; this process was dose-dependently inhibited by herceptin. The generation of VEGF-C significantly increased endothelial permeability. Pharmacological and genetic inhibition assays revealed that the cytoplasmic signaling molecule, p38 MAPK, and the transcriptional factor, NF-kappa B, are critically involved in the transcriptional activation of the VEGF-C gene by Her-2/NEU. In conclusion, this work clearly establishes that the Her-2/NEU oncogene is essential for the regulation of VEGF-C in ovarian carcinoma. It may be possible to use the monoclonal antibody targeting Her-2/NEU receptor to limit the formation of malignant ascites and lymphatic spread in ovarian carcinoma.

Inhibition of phosphorylation of a forkhead transcription factor sensitizes human ovarian cancer cells to cisplatin.

The Forkhead family transcription factor FKHRL1 is an inducer of apoptosis in its unphosphorylated form and was recently reported to be a substrate of Akt kinase. We studied the roles of FKHRL1 in both cisplatin-resistant Caov-3 (a papillary adenocarcinoma cell line) and cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of Caov-3 cells but not A2780 cells with cisplatin transiently stimulated the phosphorylation of FKHRL1. Transfection experiments revealed that a kinase inactive-mutant of Akt or a triple mutant (TM) of FKHRL1, in which all three of the putative Akt phosphorylation sites were converted to alanine, was unable to phosphorylate the FKHRL1 protein in cells treated with cisplatin. Because the phosphorylated form of FKHRL1 is known to be localized in the cytoplasm, we examined whether cisplatin-induced phosphorylation of FKHRL1 might have an effect on the subcellular distribution of FKHRL1. Cisplatin induced the localization of FKHRL1 in the cytoplasm in Caov-3 cells but not in A2790 cells. Moreover, cisplatin induced the association of 14-3-3 protein with phosphorylated-FKHRL1 in Caov-3 cells but not in A2790 cells. Because the unphosphorylated form of FKHRL1 binds the Fas ligand promoter, thereby inducing apoptosis, we further examined the effect of the phosphorylation status of FKHRL1 on the activity of the Fas ligand promoter in the presence of cisplatin. Transfection with the kinase-inactive mutant of Akt or TM of FKHRL1 induced the activity of the Fas ligand promoter in Caov-3 cells. Moreover, exogenous expression of TM of FKHRL1 in Caov-3 cells decreased the cell viability after treatment with cisplatin. Our findings suggest that cisplatin causes the phosphorylation of FKHRL1 via a phosphatidylinositol 3-kinase/Akt cascade, and inhibition of this cascade sensitizes ovarian cancer cells to cisplatin.

Secreted Ovarian Stromal Substance Inhibits Ovarian Epithelial Cell Proliferation

Objective: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. Methods: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [ 3H]thymidine uptake. Results: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in >10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. Conclusion: A heat-stable, nonheparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.

Expression and Function of β1 and αvβ3 Integrins in Ovarian Cancer

Ovarian cancer cells disseminate by implanting onto the peritoneal mesothelial cell surface of the abdominal cavity. A common feature of these peritoneal implants is the presence of tumor cell invasion into the submesothelial extracellular matrix (ECM). In view of the important role of integrins in ECM recognition and cell migration, we were interested in defining the pattern of integrin expression and function in ovarian cancer cell lines and primary tissue samples. The β1 integrin chain was expressed by CAOV-3, SKOV-3, OVCAR-3, and SW626 ovarian cancer cell lines, associated with expression of α1, -2, -3, -5, and -6 chains. The α4 chain was also expressed by two of the four lines. In addition to β1 integrins, the αvβ3 integrin was also expressed, although there was no expression of β2, -4, and -7 chains. Immunoprecipitation of surface-labeled CAOV-3 cells with an anti-β2 antibody revealed a band at ≈110-130 kDa consistent with the known molecular mass of the β1 chain, as well as several associated bands consistent with noncovalently linked integrin α chains. A similar pattern of β1 and αvβ3 integrin expression was observed for primary ovarian cancer tissue samples. Ovarian cancer cell lines exhibited significant binding to collagen type I and laminin which was primarily mediated by β1 integrins. In contrast, ovarian cancer cell binding to fibronectin was mediated by both α5β1 and αvβ3 integrins. Even though mesothelial cells were observed to express fibronectin mRNA and protein, binding of ovarian cancer cells to peritoneal mesothelium was not blocked by neutralizing antibodies to β1 or αvβ3 integrins. These data suggest that functional integrins are commonly expressed by ovarian cancer cells, although they do not appear to mediate ovarian cancer cell implantation onto peritoneal mesothelium. The role that integrins play in the invasion of ovarian cancer cells into the submesothelial ECM deserves further investigation.

Steroid Hormonal Independence of HER-2/ neu mRNA Expression in Four Human Ovarian Carcinoma Cell Lines

Overexpression of HER-2/ neu, a 185-kDa tyrosine kinase growth factor receptor, in human ovarian cancers has been correlated with a poor prognosis for survival of the disease. Previous studies have demonstrated that dexamethasone (Dex) induces a dose-dependent increase in HER-2/ neu mRNA levels in the ovarian cancer cell line SK-OV-3 by stabilizing the HER-2/ neu message. We extended these studies to test whether estrogen (Es), progesterone (Pr), and Dex were capable of regulating HER-2/ neu mRNA levels in the human ovarian cancer cell lines NIH:OVCAR-3, SW 626, OVCA 433, and Caov-3. Southern blotting demonstrated that all four cell lines contained a single copy of the 12.5-kb HER-2/ neu gene. Blotting techniques demonstrated low to barely detectable levels of HER-2/ neu mRNA and protein in these cell lines. To determine whether steroids regulated HER-2/ neu expression, all four ovarian cancer cell lines were cultured in the presence of 1 × 10 -7 M Es, Pr, or Dex and Northern blotting was completed. Unlike SK-OV-3 cells, the cell lines tested did not respond to the steroid treatments with alterations in their HER-2/ neu mRNA levels. In conclusion, neither Es, Pr, nor Dex regulates HER-2/ neu mRNA levels in NIH:OVCAR-3, SW 626, OVCA 433, and Caov-3 ovarian cancer cells. Future therapeutic manipulations of HER-2/ neu should not involve hormonal intervention.

AMPK mediates curcumin-induced cell death in CaOV3 ovarian cancer cells

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine/threonine protein kinase, serves as an energy sensor in all eukaryotic cells. Recent findings suggest that AMPK activation strongly suppresses cell proliferation and induces cell apoptosis in a variety of cancer cells. Our study demonstrated that chemopreventive agent curcumin strongly activates AMPK in a p38-dependent manner in CaOV3 ovarian cancer cells. Pretreatment of cells with compound C (AMPK inhibitor) and SB203580 (p38 inhibitor) attenuates curcumin-induced cell death. We also observed that curcumin induces p53 phosphorylation (Ser 15) and both compound C and SB203580 pretreatment inhibit p53 phosphorylation. Collectively, our data suggest that AMPK is a new molecular target of curcumin and AMPK activation partially contributes to the cytotoxic effect of curcumin in ovarian cancer cells.

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Objective: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. Methods: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. Results: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in >10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. Conclusion: A heat-stable, nonheparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.

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Objective: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. Methods: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. Results: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in >10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. Conclusion: A heat-stable, nonheparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.