Sandra S. Lawrie's research while affiliated with The University of Edinburgh and other places

The majority of ovarian cancer patients are treated with platinum-based chemotherapy, but the emergence of resistance to such chemotherapy severely limits its overall effectiveness. We have shown that development of resistance to this treatment can modify cell signaling responses in a model system wherein cisplatin treatment has altered cell responsiveness to ligands of the erbB receptor family. A cisplatin-resistant ovarian carcinoma cell line PE01CDDP was derived from the parent PE01 line by exposure to increasing concentrations of cisplatin, eventually obtaining a 20-fold level of resistance. Whereas PE01 cells were growth stimulated by the erbB receptor-activating ligands, such as transforming growth factor-alpha (TGFalpha), NRG1alpha, and NRG1beta, the PE01CDDP line was growth inhibited by TGFalpha and NRG1beta but unaffected by NRG1alpha. TGFalpha increased apoptosis in PE01CDDP cells but decreased apoptosis in PE01 cells. Differences in extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling were also found, which may be implicated in the altered cell response to ligands. Microarray analysis revealed 51 genes whose mRNA increased by at least 2-fold in PE01CDDP cells relative to PE01 (including FRA1, ETV4, MCM2, AXL, MT3, TRAP1, and FANCG), whereas 36 genes (including IGFBP3, TRAM1, and KRT4 and KRT19) decreased by a similar amount. Differential display reverse transcriptase-PCR identified altered mRNA expression for TCP1, SLP1, proliferating cell nuclear antigen, and ZXDA. Small interfering RNA inhibition of FRA1, TCP1, and MCM2 expression was associated with reduced growth and FRA1 inhibition with enhanced cisplatin sensitivity. Altered expression of these genes by cytotoxic exposure may provide survival advantages to cells including deregulation of signaling pathways, which may be critical in the development of drug resistance.

Human tumor cell lines have provided valuable model systems to study a wide variety of tumor characteristics including the cell biology, genetics, and chemosensitivity profiles of disease. A large number of ovarian cancer cell lines have now been established and are in widespread use Table 1) (1-15). Many of these have been selected to reflect specific situations, e.g., pre- and postchemotherapy models or different histo- logical subtypes. Table 1 Properties of Established Ovarian Carcinoma Cell Lines Prior Cell Line Histology Source Treatment Ref. PE01 P.D. Serous adenoca Ascites P/FU/CHL 1 PE04 P.D. Serous adenoca Ascites P/FU/CHL 1 PE06 P.D. Serous adenoca Ascites P/FU/CHL 2 PEA1 P.D. Adenoca Pleural None 2 PEA2 P.D. Adenoca Ascites P/Pred 2 PE016 P.D. Serous adenoca Ascites Radioth 2 PE014 W.D.Serous adenoca Ascites None 2 T014 W.D.Serous adenoca Sol. Met None 2 PE023 W.D.Serous adenoca Ascites P/CHL 2 SKOV-3 Adenoca Ascites T 3 SW626 Adenoca - - 3 OVCAR-2 Adenoca Ascites P/Cy 4 OVCAR-3 P.D. papillary adenoca Ascites P/Cy/Adr 5 OVCAR-4 Adenoca Ascites P/Cy/Adr 6 OVCAR-5 Adenoca Ascites None 7 OAW 28 Adenoca Ascites P / Mel 8 OAW 42 Serous adenoca Ascites P 8 41M Adenoca Ascites None 9 59M Endometr adenoca Ascites None 8 CH1 Papillary adenoca Ascites P/ JM8 8 138D Serous adenoca Ascites Carb 9 180D Adenoca Ascites P 9 200D Serous adenoca Solid None 9 253D Serous adenoca Ascites Cy/MPA 9 HOC-1 W.D. Serous adenoca Ascites None 10 HOC-7 W.D. Serous adenoca Ascites None 10 CAOV-3 Adenoca Tumour Cy/Adr/FU 10 COLO 110 Serous adenoca Sol. Met None 11 COLO 316 Serous adenoca Pleural None 11 COLO 319 Serous adenoca Ascites None 11 COLO 330 Serous adenoca Ascites Mel/Radiother 11 IGROV1 Adenoca Primary None 12 HTOA W.D. serous adenoca Primary None 13 OV-1063 Papillary adenoca Ascites Cy/Adr/P/HMM 14 DO-s W.D. mucinous adenoca Ascites - 15 P.D. = Poorly differentiated; W.D. = Well differentiated; adenoca = adenocarcinoma; pleural = pleural effusion; Sol.met. = solid metastasis; P = cisplatin; FU = 5-fluorouracil; CHL = chlorambucil; Pred = prednimustine; Radioth = radiotherapy; T = thiotepa; Cy = cyclophosphamide; Adr = adriamycin; Mel = melphalan; Carb = carboplatin; MPA = medroxyprogesterone actetate; HMM = examethylmelamine.

Langdon and coworkers (1990) have characterized a series of estrogen receptor (ER)-positive and ER-negative human ovarian carcinoma cell lines. Previous studies have demonstrated that TCDD exhibits a broad spectrum of antiestrogenic activities in ER-positive human breast cancer cell lines. Studies in this laboratory have shown that after treatment with 10 nM [3H]TCDD, specifically-bound aryl hydrocarbon (Ah) receptor complexes were identified in the cytosolic and nuclear fractions of the ER-positive PE01, PE04 and PE06 human ovarian carcinoma cell lines. Preliminary studies indicate that the molecular properties of the nuclear and cytosolic Ah receptor complexes in the ovarian carcinoma cells were similar to those reported for the Ah receptor from other human tissues and cells. The effects of TCDD on cytochrome P-4501A1 and the growth of these cell lines will be discussed.

To assess the role of oestrogen regulation in the growth of ovarian cancer, we examined the effects of an oestrogen, 17 beta-oestradiol, and an anti-oestrogen, tamoxifen, on oestrogen receptor (ER) -positive and -negative human ovarian carcinoma cell lines. As measured by a dextran-coated charcoal adsorption assay, cell lines PEO1, PEO4 and PEO6 possessed moderate concentrations of ER (96-132 fmol mg-1 protein), PEA1 and PEA2 had low values (12-23 fmol mg-1 protein) and PEO14, TO14, PEO23 and PEO16 were ER-negative. Addition of 17 beta-oestradiol (10 nM or 0.1 nM) to the ER +ve cell line, PEO4, increased the growth rate. This oestrogen stimulation could be blocked by 1 microM tamoxifen. In contrast, the growth rate of the ER -ve cell line PEO14 was unaffected by the addition of 17 beta-oestradiol or tamoxifen. Concentrations of tamoxifen in excess of 8 microM were required to produce complete cytostasis in all lines. This concentration of tamoxifen over 72 hours also inhibited 50% colony formation when cells were plated on plastic. These data indicate that some ovarian carcinoma cell lines contain ER and their growth can be sensitive to oestrogen and anti-oestrogen modulation.

We have studied the effects of sodium butyrate, retinole acid, and dimethyl sulfoxide on two human ovarian carcinoma cell lines l'I (14and PE01. PE04 cells, after treatment with sodium butyrate at cytostatic doses (2-3 HIMfor 4 days), exhibited phenotypic changes including induction of alkaline phosphatase and determinants recognized by the monoclonal antibodies 123C3 and 123A8. These effects are not simply the result of cytostasis as they were not produced by dimethyl sulfoxide or retinoic acid. Other markers are also modified by sodium butyrate including lipid, acid nimm, and glycogen. Retinoic acid modulated expres sion of lipid and CAI 25, while dimethyl sulfoxide reduced expression of CAI25. Other short chain fatty acids such as propionic acid and valeric acid (in addition to butyric acid) also induced alkaline phosphatase and the determinants recognized by 123C3 and 123A8 in PE04 cells. Other differentiation ¡mincers and cytotoxic agents studied did not induce these markers at cytostatic concentrations. The effects of sodium butyrate (and related short chain fatty acids) thus appear to be relatively specific for this cell line.

Four series of cell lines have been derived from patients with ovarian adenocarcinoma. Nine cell lines have been established at one from a solid metastasis. Six lines were derived from the ascites or pleural effusion of patients with poorly differentiated adenocarcinoma: PEO1, PEO4, and PEO6 from one patient, PEA1 and PEA2 from a second, and PEO16 from a third. Three lines (PEO14 and PEO23 from ascites and TO14 from a solid metastasis) were derived from a patient with a well-differentiated serous adenocarcinoma. Each set of cell lines was morphologically distinct. The five cell lines PEO1, PEO4, PEO6, PEA1, and PEA2 had cloning efficiencies on plastic of 1-2% and only a few cells in these lines expressed alkaline phosphatase or vimentin. Only a low percentage of these cells reacted with the monoclonal antibodies 123C3 and 123A8 but most reacted with OC125. Conversely the cell lines PEO14, TO14, PEO23, and PEO16 were characterized by low cloning efficiency values (less than 0.05%), marked expression of alkaline phosphatase and vimentin, and good reaction with 123C3 and 123A8 but not OC125. These four cell lines also exhibited dome formation. Four of the cell lines, PEO1, PEO4, PEO6, and PEO16, have been xenografted into immune-deprived mice and found to be tumorigenic.

Two ovarian cell lines were derived from the ascites of a patient before and after the onset of resistance to chemotherapy involving cis-platinum, chlorambucil and 5-fluorouracil. Characterization of these lines shows them to have various features in common and some significant differences. Cytologically the lines cannot be distinguished and they both contain high concentrations of oestrogen receptor. However, they do differ with respect to their growth characteristics, karyotype, glutathione content and sensitivity to cis-platinum. The karyotypes of the 2 lines show several marker chromosomes in common but the resistant line contained a chromosome 8 and a 17 which were absent from the earlier sensitive line. This suggests a clonal origin with subsequent divergence to a heterogeneous population.