Ovarian Tumor Cell Proliferation Research Articles

FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells.

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

The TGFβ pathway stimulates ovarian cancer cell proliferation by increasing IGF1R levels.

In a search for new therapeutic targets for treating epithelial ovarian cancer, we analyzed the Transforming Growth Factor Beta (TGFβ) signaling pathway in these tumors. Using a TMA with patient samples we found high Smad2 phosphorylation in ovarian cancer tumoral cells, independently of tumor subtype (high-grade serous or endometrioid). To evaluate the impact of TGFβ receptor inhibition on tumoral growth, we used different models of human ovarian cancer orthotopically grown in nude mice (OVAs). Treatment with a TGFβRI&II dual inhibitor, LY2109761, caused a significant reduction in tumor size in all these models, affecting cell proliferation rate. We identified Insulin Growth Factor (IGF)1 receptor as the signal positively regulated by TGFβ implicated in ovarian tumor cell proliferation. Inhibition of IGF1R activity by treatment with a blocker antibody (IMC-A12) or with a tyrosine kinase inhibitor (linsitinib) inhibited ovarian tumoral growth in vivo. When IGF1R levels were decreased by shRNA treatment, LY2109761 lost its capacity to block tumoral ovarian cell proliferation. At the molecular level TGFβ induced mRNA IGF1R levels. Overall, our results suggest an important role for the TGFβ signaling pathway in ovarian tumor cell growth through the control of IGF1R signaling pathway. Moreover, it identifies anti-TGFβ inhibitors as being of potential use in new therapies for ovarian cancer patients as an alternative to IGF1R inhibition.

Abstract B09: Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer

Abstract Sphingosine kinase-1 (SPHK1) is an enzyme that catalyzes the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the proapoptotic lipid sphingosine. The balance between sphingosine and S1P forms a sphingolipid rheostat that is primed for cell death when the balance shifts towards sphingosine/ceramide or to cell survival when S1P levels are increased. Elevated levels of S1P have been observed in several cancers, including ovarian cancer where it is elevated in both the ascites and serum of patients. Correspondingly, high expression of SPHK1 has also been described in multiple cancer types and has been linked to disease progression. Here we report that SPHK1 is overexpressed in a subset of epithelial ovarian cancers (EOC) and correlates with poor progression-free survival. Overexpression and knockdown of SPHK1 in multiple human EOC cell lines modulates in vitro cell proliferation, anchorage-independent growth, and chemosensitivity. In mouse xenograft studies, intraperitoneal administration of a SPHK1-specific inhibitor (SKI-5c) decreases tumor size, indicating that SPHK1 may be a potential therapeutic target in EOC. S1P is a secreted factor that has been shown to influence the tumor microenvironment of other cancers; however its role in EOC has not been fully established. Using a co-culture model, we show that overexpression of SPHK1 in EOC cells stimulates the transition of normal ovarian stromal fibroblasts to myofibroblasts, and enhances stromal SPHK1 expression. In a reciprocal manner, overexpression of SPHK1 in stromal fibroblast results in increased expression of MMP2 and MMP9 in EOC cells, which may contribute to a more invasive phenotype. Interestingly, stromal SPHK1 expression also enhanced expression of SPHK1 and the S1P receptor (S1PR1) in the EOC cells. Biostatistical analysis suggests a significant correlation between SPHK1 expression in EOC cells and the expression of several extracellular matrix genes (FN1, POSTN, VCAN), which have been shown to have role in the EOC tumor microenvironment. Overall, these results suggest that SPHK1 is a critical regulator of ovarian tumor cell proliferation and survival, and a mediator of tumor-stroma interaction. Citation Format: Jessica A. Beach, Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel C. Gozo, Beth Y. Karlan, Sandra Orsulic. Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B09. doi:10.1158/1538-7445.CHTME14-B09

Mechanisms of antimicrobial peptide LL-37 in macrophage-promoted ovarian cancer cell proliferation

The aim of this study was to investigate the role of macrophages in promotion of ovarian tumor cell proliferation mediated by over-expression of antimicrobial peptide LL-37. To co-culture ovarian tumor cells SKOV3, 3AO and HO-8910 with macrophages. The Transwell(®) inserts system was used in the co-culture model. The effect of macrophages promoted ovarian tumor cell proliferation was assessed by BrdU-ELISA and cell number counting. Expressions of mRNA and protein of LL-37 in the macrophages and SKOV3 cells were determined by RT-PCR and Western blot analysis. To observe that LL-37 is responsible for macrophage-promoted ovarian tumor cells growth, LL-37 neutralizing antibody was added to abrogate the LL-37 activation. The cell number assay showed that after 4 days coincubation with macrophages in the proportion of 1:0.5, the number of SKOV3 cells increased from (6.0 ± 0.5)×10(4) to (11.8 ± 1.3)×10(4), showing a significant difference (P < 0.05). It also showed that the growth of the SKOV3 cells was dependent on the macrophage number (P < 0.05). The number variability of 3AO and HO-8910 cells was as the same as SKOV3 cells upon co-culture with macrophages. As determined by BrdU-ELISA, the resulted proliferation of ovarian tumor cells was similar to the result of cell number counting. RT-PCR and Western blot results showed that the expression of LL-37 mRNA and protein in the macrophages was remarkably enhanced in a time dependent manner upon coincubation with SKOV3 cells, but did not work in SKOV3 cells. BrdU-ELISA assay exhibited that treatment of cells with LL-37 significantly stimulated HO-8910 and 3AO cell proliferation. Addition of LL-37 neutralizing antibody markedly inhibited macrophage-promoted ovarian tumor cell (SKOV3, 3AO and HO-8910 cells) proliferation. The OD values of these three cells were decreased from 2.95 ± 0.11 to 1.45 ± 0.04, from 3.39 ± 0.36 to 1.32 ± 0.09 and from 3.93 ± 0.17 to 1.68 ± 0.23, respectively (P < 0.05). Over-expression and release of LL-37 from macrophages is responsible for proliferation of ovarian tumor cells in co-culture condition. The data presented indicate that LL-37 may be critical for macrophage-induced tumor progression.

The Anticancer Ruthenium Complex KP1019 Induces DNA Damage, Leading to Cell Cycle Delay and Cell Death inSaccharomyces cerevisiae

The anticancer ruthenium complex trans-[tetrachlorobis(1H-indazole)ruthenate(III)], otherwise known as KP1019, has previously been shown to inhibit proliferation of ovarian tumor cells, induce DNA damage and apoptosis in colon carcinoma cells, and reduce tumor size in animal models. Notably, no dose-limiting toxicity was observed in a Phase I clinical trial. Despite these successes, KP1019's precise mechanism of action remains poorly understood. To determine whether Saccharomyces cerevisiae might serve as an effective model for characterizing the cellular response to KP1019, we first confirmed that this drug is internalized by yeast and induces mutations, cell cycle delay, and cell death. We next examined KP1019 sensitivity of strains defective in DNA repair, ultimately showing that rad1Δ, rev3Δ, and rad52Δ yeast are hypersensitive to KP1019, suggesting that nucleotide excision repair (NER), translesion synthesis (TLS), and recombination each play a role in drug tolerance. These data are consistent with published work showing that KP1019 causes interstrand cross-links and bulky DNA adducts in mammalian cell lines. Published research also showed that mammalian cell lines resistant to other chemotherapeutic agents exhibit only modest resistance, and sometimes hypersensitivity, to KP1019. Here we report similar findings for S. cerevisiae. Whereas gain-of-function mutations in the transcription activator-encoding gene PDR1 are known to increase expression of drug pumps, causing resistance to structurally diverse toxins, we now demonstrate that KP1019 retains its potency against yeast carrying the hypermorphic alleles PDR1-11 or PDR1-3. Combined, these data suggest that S. cerevisiae could serve as an effective model system for identifying evolutionarily conserved modulators of KP1019 sensitivity.

Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors.

BackgroundNumerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues.MethodsBreast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot.ResultsMPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines.ConclusionsMultiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

BackgroundMechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated.Methods and FindingsAt least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis.ConclusionThese findings demonstrate novel effects of γδT cells on ovarian tumor cells.

Abstract 1615: The redox modulator NOV-002 inhibits proliferation of ovarian tumor cells but increases proliferation of myeloid cells

Abstract Clinical trials in solid tumor indications have shown that NOV-002 (a formulation of glutathione disulfide) combined with standard chemotherapeutic regimens results in increased efficacy and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Non-clinical studies have interrogated the myeloproliferative and antitumor activity of the drug. NOV-002 alters redox homeostasis, at the cell surface and intracellularly, through kinase signaling culminating in differential effects on cell proliferation/survival in myeloid verses tumor cells. Thus, we previously showed that NOV-002 has positive growth effects on human myeloid lineage cells (HL60) and yet the drug leads to cell cycle arrest and apoptosis in human ovarian (SKOV3) cancer cells. In the present study, we sought to understand differences in redox-mediated signaling events that govern the opposing pharmacological properties of NOV-002. While the redox-signaling events are quite similar, the cellular consequences are distinct. Chronic treatment of cells with NOV-002 leads to enhanced proliferation in HL60 cells and growth arrest in SKOV3 cells. The alteration in growth rate occurs in both myeloid and tumor cell types in parallel with stress-induced S-glutathionylation and activation of MAP kinase pathways. In myeloid lineage cells, activation of MAP- and JAK/STAT-kinases leads to proliferation whereas activation of this pathway in SKOV3 cells leads to apoptosis. NOV-002 treatment also results in changes in plasma and mitochondrial membrane potentials in both cell models. These changes were concurrent with time- and dose-dependent increases in the accumulation of intracellular Ca2+. Interestingly, dose- and time- dependent increases in nitric oxide (NO) generation were observed in HL60 cells and shown to be mediated through eNOS. SKOV 3 cells do not express eNOS and, consequently, NO generation was not detected. Finally, redox-modulation of cell surface thiols (via S-glutathionylation) occurs in both cell types but to different extents. NOV-002 treatment leads to a ∼75% decrease in free sulfhydryls in myeloid cells whereas ovarian cancer cell surface free sulfhydryls were reduced by only ∼5%. It remains to be determined whether these observed differences in cell surface protein glutathionylation and/or in mechanism of Ca2+ flux can be linked to the opposite effects of NOV-002 on proliferation/survival in myeloid and tumor cells. Regardless of the basis of this difference, it can be speculated that the differential effects of NOV-002 on myeloid and tumor cells in vitro may relate to the unique clinical profile that NOV-002 has demonstrated to date– increased survival and anti-tumor efficacy combined with mitigation of chemotherapy-induced hematological toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1615.

Abstract 4130: Genomic profiling of ovarian tumor cell lines provides a predictive signature of sensitivity to PF-4691502, a dual PI3kinase and mTOR inhibitor

Abstract The PI3kinase/AKT/mTOR pathway plays key roles in cell growth, proliferation and survival with mTOR providing a hub for coordinating signaling in response to growth factors, hormones and nutrients. This pathway is one of the most frequently mutated or abnormally activated pathways in ovarian cancer, thus making it an excellent therapeutic target for this disease. The aims of this study were: 1. To investigate the potency of PF-4691502 (PF502) a dual PI3 kinase and mTOR inhibitor to inhibit ovarian tumor cell proliferation and induce cell death. 2. To profile exponentially growing ovarian tumor cells using gene expression, copy number variations and activation of the PI3 kinase pathway. 3. To define a predictive signature or biomarker that defines ovarian tumor cell sensitivity to PF502. A panel of 39 ovarian tumor cells were treated with PF502 (1nM-30µM) for 72h and the dose at which proliferation was inhibited by 50% (GI50) determined. Cells showed a 50-fold difference in PF502 sensitivity with GI50 values ranging from 15nM to 700nM, with the majority (85%) of cells having a GI50 of less than 200nM, these cells were classified as sensitive and the remaining 6 cell lines with a GI50 greater than 350nM were classified as resistant. PF502 also caused cell death in a subset of sensitive ovarian tumor cells. Increase in PIK3CA gene copy number was observed in 40% of the cell lines but this did not correlate with PF502 sensitivity nor did activating mutations in PIK3CA or inactivation of PTEN. Gene expression data was generated from exponentially growing cells using high-density oligonucleotide microarrays. A predictive model of response to PF502 was generated using the gene expression data. Exhaustive 10-fold cross validation of the model showed that tumour cell sensitivity could be predicted with high (96%) accuracy using approximately 20-30 genes. A differential gene expression analysis was performed using all data and 89 genes were found significantly different between sensitive and resistant cell lines. Pathway analysis revealed an over expression of inflammatory markers within the resistant class. Gene set enrichment analysis further supported an increased inflammatory signal within resistant cells, and revealed that both MYC and RAS signaling were also elevated providing a clear indication of potential targets for combination therapies in PF502 resistant disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4130.

CAMP response element–binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation

Approximately 90% of human ovarian tumors result from transformation of ovarian surface epithelial cells. It has been hypothesized that repeated destruction of the epithelial cells during ovulation, followed by proliferation and migration of epithelial cells to restore the ovarian surface, renders these cells susceptible to mutagenic events. One of the proteins found to promote ovarian surface epithelial cell survival and proliferation was the transcription factor, cAMP response element-binding protein (CREB). Thus, the objective of this study was to determine whether CREB was also highly expressed in tumor cells originating from the ovarian epithelium. Using an ovarian cancer tissue array, it was observed that approximately 54% of the epithelial-derived human ovarian tumors displayed moderate or high levels of CREB immunostaining, while none of the normal ovarian samples did. Comparison of CREB levels in a human ovarian tumor cell line to those of a normal ovarian epithelial cell line revealed elevated levels of CREB and phosphorylated CREB in the ovarian tumor cells. To determine whether CREB regulated proliferation and/or apoptosis in the ovarian tumor cell line, CREB expression was suppressed using RNA interference. Decreased CREB expression significantly reduced ovarian tumor cell proliferation, while there was no effect on apoptosis in these cells. Finally, we showed that CREB is highly expressed in an in vivo murine model of ovarian tumorigenesis. Therefore, CREB is frequently overexpressed in ovarian cancer where it appears to promote cell proliferation.

Cell proliferation activity unrelated to COX-2 expression in ovarian tumors

The objective of this study was to assess the expression of Cyclooxygenase-2 (COX-2) and cell proliferation activity (Ki67 expression) in benign, borderline, and malignant serous and mucinous ovarian tumors. Expression of COX-2 and Ki67 proteins were evaluated by immunohistochemistry, in paraffin-embedded sections of ovarian epithelial tumors. The study included 113 serous (67 benign, 15 borderline, and 31 malignant) and 85 mucinous (48 benign, 28 borderline, and 9 malignant) tumors, removed from women who underwent laparotomy between January 1997 and December 2003. From benign to malignant tumors, there was a progressive positive trend in COX-2 expression in both serous and mucinous tumors, more evident in mucinous ones (P < 0.001). Comparing histologic types, COX-2 expression was more prominent in serous than in mucinous benign tumors (P < 0.01), but this difference was not significant in the borderline (P= 0.11) or malignant categories (P= 0.71). There was a progressive Ki67 positivity in line with the tumor histologic gradient for both serous (P < 0.01) and mucinous lesions (P < 0.01), but this increasing expression did not correlate with COX-2 expression in the present series (P= 0.78). There was a higher COX-2 expression in serous ovarian adenomas than in mucinous ones. COX-2 positivity increases in line with the morphologic gradient, from benign to malignant in both histologic types, but it was more prominent in mucinous lesions, pointing to different oncogenic pathways related to different histologic types. A correlation between the expression of COX-2 and Ki67 was not found, suggesting that COX-2 may be required for carcinogenesis, but this pathway is not responsible for cell proliferation in ovarian tumors.

Human DNA topoisomerase II-alpha

DNA topoisomerase II-alpha (topo II-alpha) is the molecular target of several types of clinically useful anticancer drugs such as etoposide, teniposide, doxorubicin, and mitoxantrone. The enzyme is also a proliferation marker in normal cells and tissues. High levels of enzyme are present in the S and G2 phases of the cell cycle. New data are emerging which suggest that anticancer drugs which target the topoisomerases may be useful in the treatment of patients with ovarian neoplasms. However, there is relatively little data on the expression of these enzymes in human ovarian cancer. We have recently developed an in situ immunohistochemical stain which can detect the presence of topo II-alpha in formalin-fixed paraffin-embedded human tissue sections. In order to determine topo II-alpha levels in ovarian tumors, we immunostained for topo II-alpha, 30 ovarian neoplasms which ranged from benign to highly malignant. We correlated our results with expression of MIB1 in order to determine if topo II-alpha expression correlates with cell proliferation. Since the gene for topo II-alpha is closely linked to the gene for the c-erbB-2 oncogene, we also evaluated the cases for amplification of c-erbB-2. Our results indicate that topo II-alpha correlates well with MIB1 indicating that topo II-alpha may be useful in estimating cell proliferation in ovarian tumors. In addition, 1 of 15 patients with a malignant neoplasm had a carcinoma which expressed high levels of topo II-alpha. Since the sensitivity of cells to topo II targeted drugs is dependent on high topo II levels, this suggests that topo II-alpha immunostaining in ovarian cancer may also identify a subset of patients potentially treatable with topo II targeted drugs. None of the ovarian neoplasms showed amplification of c-erbB-2.

Molecular approaches to prevention and detection of epithelial ovarian cancer.

More than 90% of epithelial ovarian cancers arise from single cells. Malignant transformation can be associated with a number of molecular alterations including upregulation of tyrosine kinases and phosphatases, physiologic activation o ras, mutation of p53, amplification of myc, and increased activity of matrix metalloproteinases 2 and 9. Proliferation of transformed epithelial cells can be enhanced through the persistence of autocrine growth stimulation by TGF-alpha, loss of autocrine growth inhibition by TGF-beta, as well as paracrine growth stimulation by macrophage derived cytokines and OCAF, a novel lyso-phospholipid. Ascites tumor cells retain responsiveness to growth inhibition by TGF-beta which induces apoptosis in malignant ovarian epithelial cells, but not in normal ovarian surface epithelium. Proliferation of surface epithelial cells following ovulation may contribute to the pathogenesis of ovarian cancer. Use of oral contraceptives that suppress ovulation has been associated with reduced risk of ovarian cancer in later life. Retinoids also deserve further evaluation for chemoprevention. Treatment with fenretinide was associated with decreased incidence of ovarian cancer. Additive or synergistic inhibition of ovarian tumor cell proliferation has been observed with TGF-beta in combination with all-trans-retinoic acid. Early detection of ovarian cancer could improve survival. Transvaginal sonography (TVS) and serum markers such as CA-125 have been evaluated in multiple clinical trials. The former lacks adequate specificity, whereas the latter is not sufficiently sensitive. Use of multiple serum markers can improve sensitivity. A combination of CA-125, M-CSF and OVX-1 has detected > 95% of Stage I ovarian cancers. If similar results are obtained with different data sets, multiple serum markers could be used to trigger the performance of TVS, providing a potentially cost effective screening strategy. Prospective trials will be required to demonstrate that screening for early stage ovarian actually impacts on survival.

Stimulation of ovarian tumor cell proliferation with monocyte products including interleukin-1, interleukin-6 and tumor necrosis factor-α

Stimulation of ovarian tumor cell proliferation with monocyte products including interleukin-1, interleukin-6 and tumor necrosis factor-α

Stimulation of ovarian tumor cell proliferation with monocyte products including interleukin-1, interleukin-6, and tumor necrosis factor-α

We investigated whether monocyte-derived factors could stimulate the growth of ovarian cancer cells. Human peripheral blood monocytes or human monocyte-like cell lines THP-1 and U-937 were cultured with or without macrophage colony-stimulating factor, lipopolysaccharide, or phorbol myristate acetate. Culture supernatants or recombinant cytokines were assayed for growth stimulation of ovarian cancer cell lines by tritium-thymidine incorporation and direct cell counts followed by statistical analysis with Student t test. Conditioned medium from peripheral blood monocytes or from THP-1 or U-937 cells stimulated ovarian cancer cell growth. Interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6 also stimulated ovarian cancer cell growth, whereas macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factor did not. Concentrations of tumor necrosis factor, interleukin-1, and interleukin-6 in conditioned medium could not account for all the growth stimulation, and activity remained after neutralization of tumor necrosis factor, interleukin-1, and interleukin-6 with antibodies. Interleukin-1, interleukin-6, tumor necrosis factor, and additional monocyte factor(s) could provide paracrine growth stimulation when monocytes are attracted to ovarian cancers that produce macrophage colony-stimulating factor.

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.