The role of interleukin-1 in interactive senescence and age-related human endometrial cancer.

The causes of the age-related increase in cancer rates are poorly understood. One cause could be age-related changes in the stromal/epithelial cell interactions that facilitate tumorigenesis. We tested the hypothesis that aging of human endometrial stromal fibroblasts (ESF) alters their influence over endometrial epithelial cells. ESF from adults were found to inhibit anchorage-independent proliferation, to restrain colony outgrowth, and to induce formation of normal tissue architecture by human endometrial cancer cells. As ESF age, these inhibitory influences on malignant-like behaviors by epithelial cells are altered, becoming stimulatory. Age-related change in interleukin-1alpha (IL-1alpha) expression is a molecular determinant of ESF/epithelial cell interactions. Levels of IL-1alpha and IL-1-induced mRNAs increase in ESF with age. Treatment with IL-1 accelerates age-related changes in mRNA abundance and loss of ESF restraint over malignancy-associated behaviors by epithelial cells. Transfection of ESF with the intracellular IL-1 receptor antagonist preserved the young phenotype with respect to interactions with epithelial cells and prevented age-associated increases in groalpha and IL-8 mRNA levels. Our results indicate that aging of ESF is accompanied by an interactive senescence that alters ESF signaling to cancer cells and could contribute to increased cancer rates by providing a microenvironment that is more conducive to tumorigenesis.

Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence.

Aging is the major risk factor for many cancers, and age-related changes in the tissue microenvironment can facilitate tumor growth. This study uses human endometrial cells to begin to test the hypothesis that age-related changes in pigment epithelium-derived factor/early population doubling cDNA-1 (PEDF/EPC-1) levels create an environment that is more permissive to tumor growth. Endometrial stromal fibroblasts (ESF) are the predominant cell type in the human endometrium and exert regulatory control over the glandular epithelial cells, which are the source of most tumors. As ESF age in vitro, their ability to regulate appropriate growth and differentiation of epithelial cells declines. Endometrial epithelial cells in primary culture expressed relatively low levels of PEDF/EPC-1 mRNA. In contrast, early passage quiescent ESF from adult donors produce higher levels of the 1.5-kb PEDF/EPC-1 mRNA and 50-kDa secreted protein than epithelial cells. As ESF age in vitro the relative abundance of PEDF/EPC-1 mRNA declines, as does the level of PEDF/EPC-1 protein secreted into cell culture medium. Treatment with PEDF/EPC-1 protein had no effect on ESF proliferation but did inhibit anchorage-dependent and anchorage-independent proliferation of endometrial carcinoma cells in a dose- and time-dependent manner. These findings imply that an age-related loss of PEDF/EPC-1 expression by ESF could eliminate a negative regulator of cancer cell growth and, thereby, contribute to the age-related increase in cancer incidence.

Regulation of keratinocyte growth factor expression in human endometrium: implications for hormonal carcinogenesis.

Estrogen is thought to be an important etiologic agent in endometrial and breast cancers. However, the mechanism or mechanisms by which estrogen acts as a hormonal carcinogen are not well understood. We hypothesize that in response to chronic exposure to estrogens, human endometrial stromal fibroblasts (ESF) produce factors that facilitate neoplastic transformation in epithelial cells. To test this hypothesis, we assessed the regulation of keratinocyte growth factor (KGF) mRNA and protein in ESF by interleukin-1 (IL-1) and diethylstilbestrol (DES). Short-term treatments with IL-1 but not with DES increased the abundance of KGF mRNA in ESF. However, chronic treatment with DES significantly increased KGF mRNA levels and protein production. KGF protein in medium conditioned by ESF chronically treated with 1 nM DES reached concentrations of approximately 100 ng/mL. At this concentration, KGF increased endometrial epithelial cell numbers fourfold and enhanced anchorage independence tenfold. These results suggest that KGF may play a role in hormonal carcinogenesis by mediating estrogen-induced changes in the interactions between stromal and epithelial cells. To address the potential role of nuclear transcription factor kappa B (NF-kappaB) in regulating KGF expression, we determined the effect of increased expression of its inhibitor, IkappaBalpha, on KGF mRNA and protein levels. Transfection with IkappaBalpha blocked induction of KGF expression by IL-1 but had no effect on the increase in KGF mRNA caused by chronic treatment with DES. These results suggest that IL-1 exerts its effects on KGF by an NF-kappaB-mediated pathway but that chronic treatment with DES stimulates KGF expression by some other mechanism.

Selective potentiation of gynecologic cancer cell growth in vitro by electromagnetic fields.

OBJECTIVE: Epidemiological data suggest that exposure to electromagnetic fields (EMF) may increase the risk of various cancers. We evaluated EMF effects on the in vitro growth response of human cell lines isolated from various reproductive tract tissues. We also assessed the effects of EMF on cisplatin- or paclitaxel-induced cytotoxicity. METHODS: Endometrial, ovarian, and prostate cancer cell lines as well as immortalized endometrial stromal cells and immortalized ovarian epithelial cells were exposed continually to EMF. Proliferation was assessed by the metabolic activity assay, MTT, direct cell counting, and anchorage-independent colony formation in soft agar. Cytotoxicity induced by cisplatin or paclitaxel was assessed using the MTT assay. RESULTS: Continuous exposure to EMF at field strengths of 2 G enhanced proliferation of two human prostate and three endometrial, but only one ovarian, cancer cell lines. EMF enhanced metabolic activity of cancer cells within 96 h and increased absolute cell number (anchorage-dependent proliferation) and colony-forming efficiency (anchorage-independent proliferation) over sham-treated controls. EMF had no effect on cytotoxicity induced by the chemotherapeutic agents Taxol or cisplatin. CONCLUSIONS: Continuous exposure to EMF can enhance growth rates of transformed cells for some human epithelial cancers. Cancer cells from the steroid sex hormone regulated tissues of endometrium and prostate appeared to be more responsive to EMF than cells from ovarian cancers.

Identification of the structural region of taxol that may be responsible for cytokine gene induction and cytotoxicity in human ovarian cancer cells.

PURPOSE: Interleukin-8 (IL-8) is a pleiotropic chemokine with both chemoattractant and angiogenic properties. In addition to its cytotoxic effects on ovarian cancer cells, taxol can transcriptionally activate genes such as IL-8 that may play a role in tumorigenesis. Utilizing IL-8 as a prototypic marker of tumor-derived modulators of growth, we undertook a systematic study of taxol and 11 structurally modified taxol analogs to identify the region of the taxane skeleton responsible for IL-8 gene induction. METHODS: The human ovarian cancer cell line OVCA-420 was exposed to taxol or taxol analogs. IL-8 gene induction was assessed by Northern blot analysis after 6 h and cytotoxicity after 72 h. RESULTS: Changes in the southern hemisphere (C-1 to C-4) of the taxane skeleton had greater effects on IL-8 induction than changes in the northern hemisphere (C-7 to C-11). Some of the taxol analogs modified at positions C-1 and/or C-2 with increased hydrophobicity induced IL-8 expression more than threefold over that induced by taxol or taxotere and more than 20-fold over control cells. Cells that failed to induce IL-8 gene expression in response to taxol were only marginally responsive to the analogs unless first primed with IL-1beta. Modifications to the northern hemisphere did not alter taxol's effect on IL-8 expression in human cells, but did influence TNFalpha expression in murine macrophage cells, suggesting species and/or gene specificity. We found a direct correlation between IL-8 induction and cytotoxicity, in that analogs that dramatically upregulated IL-8 expression proved to be the most cytotoxic, inhibiting cell survival by > 90%. CONCLUSION: Taken together our results demonstrate that changes in the southern hemisphere of the taxane skeleton influence both the gene induction and cytotoxic potential of taxol in human ovarian cancer cells.

Diethylstilbestrol-induced immortalization of human endometrial cells: alterations in p53 and estrogen receptor.

Carcinogenesis is a process requiring multiple steps. Immortalization is one step in this process and may be rate limiting. To further our understanding of estrogen-induced carcinogenesis, we evaluated diethylstilbestrol (DES)-induced immortalization of human endometrial stromal cells. This was achieved by assessing at the restrictive temperature the colony-forming efficiency of cells that were conditionally immortalized with a temperature-sensitive simian virus 40 large T antigen. Treatment with DES for 1 wk did not increase the immortalization frequency; however, cultures that were treated for 20 wk had a twofold increase in immortalization frequency, and continued treatment for a total of 44 wk produced a threefold increase in immortalization frequency that was dose dependent. DES-treated restrictive temperature variants (RTVs) but not spontaneous RTVs lost the temperature-sensitive phenotype. DES-RTVs also had a shorter doubling time than spontaneous RTVs did. p53 expression was increased in DES-RTVs, and its localization within the cell was altered. Conversely, expression of the estrogen receptor was decreased in DES-immortalized cells. These changes in gene expression often occur in estrogen-related malignancies, and our results are consistent with a causal role for estrogens in these p53 and the estrogen receptor alterations. Immortalization of human cells may be analogous to initiation of rodent cells, and our results suggest that estrogen-induced alterations in p53 or other genes that regulate life span could contribute to estrogen-induced initiation.

Basement membrane induced differentiation of HEC-1B(L) endometrial adenocarcinoma cells affects both morphology and gene expression.

In vitro studies of endometrial carcinogenesis have been hampered by dedifferentiation of the cells in culture. Using the endometrial carcinoma cell line HEC-1B(L), we aimed to establish and characterize culture conditions that preserve a more differentiated state of the tumor cells. HEC-1B(L) cells grown in a serum-free defined medium on plastic (PL/SFDM) on top of a reconstituted basement membrane (Matrigel, MG/SFDM) or in a thick layer of Matrigel showed pronounced morphological differentiation as compared with HEC-1B(L) cells cultured on plastic in a medium containing serum (PL/10% FCS). Features of differentiation included cuboidal to columnar cell shape and an increase of rough endoplastic reticulum in Matrigel cultures. Gene expression of HEC-1B(L) cells was studied by metabolic [35S]methionine labeling and SDS-gel electrophoresis. HEC-1B(L) cells cultured in the presence of Matrigel showed two additional secretory proteins approximately 31 kD and 77 kD in size. rt-PCR was used to screen cell cultures for the presence of estrogen receptor, progesterone receptor, and lactoferrin-mRNA, genes typically expressed by normal endometrial epithelium. We found no expression of the estrogen receptor or progesterone receptor. Lactoferrin-mRNA was present under all culture conditions tested. Our results suggest a regulatory role of the extracellular matrix for the differentiation of the HEC-1B(L) cell line.

Tumor cell invasion of basement membrane in vitro is regulated by amino acids.

Because most cancer deaths result from disseminated disease, understanding the regulation of tumor invasion and metastasis is a central theme in tumor cell biology. Interactions between extracellular matrices (ECM) and cellular microenvironment play a crucial role in this process. We have tested selected amino acids and polyamines for their ability to regulate RL95-2 cell invasion through both intact human amniotic basement membrane and a novel human ECM (Amgel). Three major systems for neutral amino acid transport, systems L, A, and ASC, are operational in these neoplastic cells. Amino acids entering the cell via transport system A or N, i.e., (methyl amino)-isobutyrate (MeAIB) or Asn, markedly enhanced invasiveness of these human adenocarcinoma cells as measured by a standard 72-hr amnion or Amgel invasion assay. Addition of 2-amino-2-norborane carboxylic acid (BCH; 1 mM), a model substrate of the L transport system, caused a significant decrease in invasive activity when tested in the Amgel assay. Interestingly, Val lowers steady-state levels of MeAIB uptake and blocks the increase in cell invasion elicited by MeAIB. At the same time, these amino acids do not influence cell proliferation activity. Neither the charged amino acid Lys or Asp (not transported by A/N/L systems) nor the polyamines putrescine, spermidine, or spermine modulate invasiveness under similar experimental conditions. Moreover, the observed time-dependent stimulation of system A activity (cellular influx of MeAIB) by substrate depletion is prevented by the addition of actinomycin D (5 microM) or cycloheximide (100 microM), suggesting the involvement of de novo RNA and protein synthesis events in these processes. MeAIB treatment of tumor cells selectively increased the activities of key invasion-associated type IV collagenases/gelatinases. These results indicate that in the absence of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumor cell invasion and, by extension, metastasis. We propose that amino acids, acting via specific signaling pathways, modulate phenotypic cell behavior by modulating the levels of key regulatory enzymatic proteins.

The intracellular IL-1 receptor antagonist alters IL-1-inducible gene expression without blocking exogenous signaling by IL-1 beta.

The epithelium-associated tissue distribution of the intracellular IL-1R antagonist (icIL-1Ra) suggests that it functions as a novel regulatory molecule for IL-1 in somatic tissues. We examined the role of the icIL-1Ra in IL-1 beta-induced responses in human ovarian cancer cells because ovarian surface epithelium expresses transcripts for the icIL-1Ra, and the majority of ovarian cancers arise from these cells. Several human ovarian and cervical cancer cell lines spontaneously express the icIL-1Ra. icIL-1Ra-expressing cells did not have altered growth characteristics or altered short term responses to IL-1 compared with icIL-1Ra-nonexpressing cells. While a 90-min exposure to IL-1 beta resulted in increased steady state cytokine mRNA levels in all cells, icIL-1Ra-positive cells were incapable of maintaining IL-1-beta-induced expression of GRO mRNA. This did not result from decreased transcriptional activity of the GRO gene, but reflected differences in mRNA stability and/or degradation. To determine whether the icIL-1Ra altered mRNA stability, we used a retroviral expression vector to express the icIL-1Ra in an icIL-1Ra-negative cell line. The resulting cells displayed a profile of IL-1 beta-induced genes analogous to that found in cells spontaneously expressing icIL-1Ra. These data show for the first time an intrinsic biologic activity for the icIL-1Ra. The capacity to selectively alter IL-1-induced gene expression suggests that this version of the IL-1Ra is a unique intracellular inhibitor that attenuates IL-1 responses at a point downstream of the initial IL-1/IL-1 receptor interaction.

Estrogen-induced anchorage-independence in human endometrial stromal cells.

Estrogens are important etiologic agents for most gynecologic malignancies, and chronic exposure to estrogen that is unopposed by progestins conveys the greatest risk. Treatments with estrogen facilitate the process of malignant transformation in rodents, but relatively few studies of estrogen-induced carcinogenesis have been performed using human cells. Most malignancies in estrogen-responsive tissues arise from epithelial cells, but an increasing body of evidence emphasizes the role of stromal cells as mediators of the effects of estrogens on epithelial cells. Our studies were designed to assess estrogens as carcinogens for human endometrial stromal cells and to provide a basis for studies of the role of stroma in estrogen-induced carcinogenesis in humans. Acute treatments with the estrogens diethylstilbestrol (DES), 17 beta-estradiol (E2) and beta-dienestrol enhance anchorage-independent proliferation (AIP) of SV40-immortalized human endometrial stromal cells in the rank order of DES > E2 > beta-dienestrol. The anti-estrogenic compound tamoxifen inhibits DES-induced AIP. The magnitude of DES-induced AIP increases with prolonged duration of treatment. After 11 months of chronic treatment with 0.1 nM DES, AIP was 20-fold higher than in vehicle-treated control cultures. Expression of the estrogen receptor was altered by treatments with DES in parallel with increased capacity for AIP. These conditionally immortal human endometrial stromal cells appear to be a good model for estrogen-induced transformation of human cells.

Productive infection of human endometrial stromal cells by human cytomegalovirus.

Cultured endometrial stromal cells were susceptible to productive human cytomegalovirus (HCMV) infection. Infection of endometrial stromal cells resulted in pronounced cytopathic effects including cell rounding and aggregation, fusions, and some lysis, although not in the synchronous fashion observed in infected fibroblasts. The aggregation events were reminiscent of normal endometrial stromal cell responses to cyclical estrogen/progesterone levels. Immunofluorescence analysis demonstrated expression of viral gene products suggesting a productive virus infection. One-step growth analysis showed that infectious virus was produced but the titers were two logs lower than those obtained in fibroblasts even though HCMV DNA accumulated to similar levels in both cell types. In contrast, viral DNA replication was greatly reduced in endometrial stromal cells immortalized with a temperature-sensitive SV40 large T gene at both permissive and nonpermissive temperatures. A more detailed analysis of viral gene expression by Northern blotting revealed earlier appearances and greater initial levels of viral transcripts in endometrial stromal cells. No HCMV gene expression was observed at 120 hpi in these cells even though half of the cells were still intact and cellular gene expression was functional. Since this was the time of peak virus production, it seems plausible that reduced viral gene expression at late times p.i. was a major contributor to the reduced titers observed in endometrial stromal cells. These in vitro results coupled with in vivo observations by others of endometritis associated with HCMV suggest that further investigation into the effects of HCMV on the endometrium is warranted.

In vitro interactions of endometrial stromal and epithelial cells in Matrigel: reorganization of the extracellular matrix.

Fragments of human endometrial glands and dispersed endometrial stromal cells were cultured together in a thick layer of reconstituted basement membrane (Matrigel). Epithelial cells kept their glandular morphology whereas stromal cells grew into round clusters of mainly fusiform cells. Transmission electron micrographs showed collagen fibers between stromal cells as well as in surrounding extracellular matrix after 2.5 weeks. A well-defined basement membrane was found when epithelial and stromal cells were in close proximity to each other. Beneath the lamina densa there was a loose network of collagen fibers or a dense fibrillar network arranged parallel to the cell layers. Epithelial cells showed hemidesmosomes at their basal surface where they were close to stromal cells.

H-ras codon 12 mutation in cervical dysplasia.

A mutation in codon 12 of the H-ras oncogene has been implicated in the pathogenesis of cervical cancer. To determine if mutational activation of H-ras is an early event in carcinogenesis, we looked for the presence of codon 12 H-ras mutation in precancerous cervical dysplasia. We analyzed cervical DNA from 63 normal patients and 72 patients with biopsy-proven cervical dysplasia [CIN I (48), CIN II (9), CIN III (15)]. HPV typing was performed on these samples by Viratype and analysis and polymerase chain reaction (PCR). To detect H-ras codon 12 mutation, DNA was isolated and amplified by PCR with primers for H-ras. A unique restriction site for MspI contained within the wild-type ras PCR product allows discrimination between unmutated and mutated sequences. Restriction enzyme analysis with MspI was performed on PCR products to distinguish between wild-type and mutated sequences. HPV 16 or 18 was present in 33% of CIN I, 56% of CIN II, and all CIN III samples. No codon 12 mutations were detected in any sample. We conclude that while HPV is associated with cervical dysplasia and its prevalence increases in more advanced lesions, it is unlikely that H-ras codon 12 mutations are an early occurrence in the progression of precancerous lesions to cervical carcinoma.

Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40.

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.

Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.

Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines. Of the 11 CF clones isolated, 6 developed a polarized phenotype with vectorial ion transport and membrane-specific expression of histamine and purinergic receptors. The ion transport properties of these lines differed from the normal lines and approximated those of primary CF airway epithelial cell cultures more closely than do those of cell lines transformed with the simian virus 40 large T gene. When transplanted into denuded tracheal grafts, these cells can differentiate into ciliated and secretory phenotypes. We conclude that HPV-18 E6 and E7 genes are sufficient to transform human airway epithelial cells and that the resultant cell lines express differentiated phenotypic properties that approximate those of the native epithelium.

Development of a novel human extracellular matrix for quantitation of the invasiveness of human cells.

During the crucial stages of tumor cell invasion and metastasis, neoplastic cells must traverse extracellular matrices for their migration to distant sites. Because basement membranes (BM) serve as a critical barrier to such passages, most previous in vitro assay models have utilized either an intact BM or a reconstituted rodent or avian BM-matrix to study this process. We have created a gel-like extracellular matrix derived from human amnions which contained type IV collagen, laminin, entactin, tenascin and heparan sulfate proteoglycan. This matrix, which we called Amgel, was used to study selected steps of invasion including cell attachment to matrix, degradation of it by proteolytic enzymes and movement of human tumor cells through matrix defects. An efficient tumor invasion assay system was developed utilizing filter-supported uniform coatings of this matrix in chambers. Human tumor cells (HT-1080 fibrosarcoma and RL-95 adenocarcinoma), when seeded onto Amgel-coated membranes, attached to matrix within 2 h and initiated a time-dependent migration and invasion process, as verified by biochemical analysis and both light and electron microscopy. In an optimized invasion assay 12-15% of tumor cells completely traversed the matrix during a 72-h period with > 90% viability. In contrast to these highly-invasive cells, normal human foreskin fibroblasts and normal human endometrial stromal cells exhibited minimal migration/matrix penetration during the same time period. When the Amgel-selected tumor cells (i.e. those penetrating the barrier) were isolated, subcultured, and re-exposed to Amgel, they had heightened invasiveness (2-3-fold) as compared to the parental cells. Thus, this improved 'all human' system for quantitating the invasive ability of tumor cells may provide a valuable tool in dissecting out the mechanistic underpinnings of human metastasis. In addition, this assay has the ability to screen agents which have potential anti-invasive and by extension anti-metastatic, activity or chemotactic properties.

Expression of tissue kallikrein in normal and SV40-transfected human endometrial stromal cells.

The polymerase chain reaction with specific tissue kallikrein primers was utilized to demonstrate the presence of tissue kallikrein mRNA in human endometrial stromal cells. Enzymatic analysis measured with a specific tripeptide nitroanilide substrate demonstrated the presence of tissue kallikrein in the conditioned medium obtained from both normal stromal cells and stromal cells transfected with an origin-defective temperature-sensitive SV40 large T antigen. The transfected stromal cell supernatant exhibited approximately twice as much tissue kallikrein activity as normal stromal cells at 60-100% of cell confluence. The release of tissue kallikrein from transfected stromal cells was confirmed by Western blot analysis and [35S]-methionine incorporation into a 35-kD protein which retains tissue kallikrein activity. These results demonstrate for the first time the expression and secretion of tissue kallikrein in human endometrial stromal cells and provide evidence of possible involvement of tissue kallikrein in cell transformation.

Interleukin-1 inhibits growth of normal human endometrial stromal cells.

OBJECTIVE: To understand growth regulation of the endometrium by studying the effect of interleukin-1 beta (IL-1) on human endometrial stromal cell proliferation in vitro. METHODS: Endometrial stromal cells from human endometrium were separated and purified and placed in culture. Fresh and first- and sixth-passage cells were incubated with IL-1 (0.025, 0.25, and 2.5 ng/mL) for 24, 48, and 72 hours, respectively. Proliferation as a function of DNA synthesis was assessed by measuring 3H-thymidine incorporation. Experiments were then repeated in the presence of indomethacin to determine whether IL-1 effects were dependent upon prostaglandin synthesis. We evaluated overall growth by adding IL-1 to cell cultures of sixth-passage stromal cells every 3 days and by performing cell count studies. RESULTS: Interleukin-1 beta significantly inhibited 3H-thymidine uptake in freshly explanted endometrial stromal cells at all doses in a dose-dependent manner; a 44% inhibition was seen at 2.5 ng/mL IL-1 after 72 hours of incubation. In first- and sixth-passage cells, 3H-thymidine uptake was inhibited only at intermediate and high doses of IL-1. Cell count studies showed that sixth-passage cells were significantly inhibited by IL-1 after 23 days of growth (22%; P less than .01). Adding indomethacin did not affect inhibition of growth. CONCLUSION: Interleukin-1 beta inhibits growth of normal human endometrial stromal cells in vitro and does not appear to be mediated by arachidonic acid metabolites. This inhibition of growth may be important for maintenance of a normal endometrial phenotype.

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.