BARX2 induces cadherin 6 expression and is a functional suppressor of ovarian cancer progression.

The human homeobox BARX2 is located at 11q24-q25, within a minimal region associated with frequent loss of heterozygosity and adverse survival in epithelial ovarian cancer. BARX2 is a transcription factor that regulates transcription of specific cell adhesion molecules in the mouse. We show that BARX2 and cadherin 6 are expressed in normal human ovarian surface epithelium. BARX2 and cadherin 6 both have significantly lower expression in a clinical sample of endometrioid and clear cell ovarian cancers, as compared with serous or mixed mesodermal tumors. In a series of ovarian cancer cell lines, BARX2 expression showed a significant direct correlation with cadherin 6 expression. In OAW42, an ovarian cancer cell line that does not endogenously express BARX2, in vitro transfection of human BARX2 cDNA induced cadherin 6 expression. Transfection of BARX2 into OAW42 inhibited Matrigel invasion, haptotactic cellular migration to a collagen IV signal, and adhesion to collagen IV-coated plates. Our data demonstrate that BARX2 is expressed in the ovarian surface epithelium and has functional suppressor properties in ovarian cancer cells.

Differential display.

Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) is an extremely powerful method for analyzing differences in gene expression between matched tissue/cell samples. Liang and Pardee originally described the technique in 1992 in their seminal paper (1). DDRT-PCR is now firmly established as a widespread powerful and commonly used method for identifying differentially expressed genes.

Identification of a region of frequent loss of heterozygosity at 11q24 in colorectal cancer.

Loss of heterozygosity (LOH) at 11q23-qter occurs frequently in ovarian and other cancers, but for colorectal cancer, the evidence is conflicting. Seven polymorphic loci were analyzed between D11S897 and D11S969 in 50 colorectal tumors. Two distinct LOH regions were detected, suggesting possible sites for tumor-suppressor genes involved in colorectal neoplasia: a large centromeric region between D11S897 and D11S925, and a telomeric 4.9-Mb region between D11S912 and D11S969. There was no correlation with clinicopathological features. This analysis describes a region of LOH in the region 11q23.3-24.3 for the first time in colorectal cancer and provides complementary evidence for the ongoing effort to identify the gene(s) involved.

Inhibition of transforming growth factor alpha (TGF-alpha)-mediated growth effects in ovarian cancer cell lines by a tyrosine kinase inhibitor ZM 252868.

The modulating effects of the epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor ZM 252868 on cell growth and signalling have been evaluated in four ovarian carcinoma cell lines PE01, PE04, SKOV-3 and PE01CDDP. Transforming growth factor alpha (TGF-alpha)-stimulated growth was completely inhibited by concentrations > or =0.3 microM in the PE01 and PE04 cell lines and by > or =0.1 microM in SKOV-3 cells. TGF-alpha inhibition of PE01CDDP growth was reversed by concentrations > or =0.1 microM ZM 252868. TGF-alpha-stimulated tyrosine phosphorylation of both the EGF receptor and c-erbB2 receptor in all four cell lines. The inhibitor ZM 252868, at concentrations > or =0.3 microM, completely inhibited TGF-alpha-stimulated tyrosine phosphorylation of the EGF receptor and reduced phosphorylation of the c-erbB2 protein. EGF-activated EGF receptor tyrosine kinase activity was completely inhibited by 3 microM ZM 252868 in PE01, SKOV-3 and PE01CDDP cells. These data indicate that the EGF receptor-targeted TK inhibitor ZM 252868 can inhibit growth of ovarian carcinoma cells in vitro consistent with inhibition of tyrosine phosphorylation at the EGF receptor.

Identification and characterization of a homozygous deletion found in ovarian ascites by representational difference analysis.

We have performed representational difference analysis (RDA) on DNA from tumor cells and normal fibroblasts isolated from the ascites of a patient with ovarian cancer. Five of six products of the RDA were homozygously deleted from the tumor DNA. One of these products has been characterized and identifies a homozygous deletion of approximately 6.9 Mb at chromosome 9p21 in the original ovarian tumor material. This deletion encompasses CDKN2A (p16), CDKN2B (p15), and IFN-alpha. PCR analysis of other tumor cell lines using the novel STS based on the RDA product has shown it to lie between IFN-alpha and p16, and to identify the distal extent of a homozygous deletion in another ovarian cancer cell line. These data provide further evidence for a tumor suppressor locus distinct from, but mapping close to, p16 on 9p21. Cytogenetic analysis using comparative genomic hybridization (CGH) performed on the same primary tumor confirmed a loss of material from chromosome 9p. However, the CGH technique had neither the resolution nor the sensitivity to define a subregion of homozygous loss.

Growth-inhibitory effects of the synthetic retinoid CD437 against ovarian carcinoma models in vitro and in vivo.

The activity of CD437¿6-[3-(1-adamantyl)-4 hydroxyphenyl]-2-naphthalene carboxylic acid¿, a relatively selective activator of RAR-gamma, was evaluated against four human ovarian-carcinoma cell lines : PE01, PE04 (a Pt-resistant in vivo-derived counterpart of PE01), PE01CDDP (a Pt-resistant in vitro-derived model of PE01) and PE014. Growth inhibition was observed after 3 and 6 days of exposure to sub-micromolar concentrations as assessed by a reduction in cell number. IC50 values against PE01, PE04, PE01CDDP and PE014 were 0.09, 0.21, 0.12 and 0.28 microM (day 3) and 0.1, 0.14, 0.07 and 0.17 microM (day 6), respectively. Cisplatin-resistant cell lines were as responsive as cisplatin-sensitive lines, indicating potential activity in resistant disease. CD437 was also evaluated against the PE04 xenograft grown in nude mice using daily doses of 20 (days 0-4) and 10 mg/kg (days 0-4 and 7-11) given either by i.p. delivery or oral administration. Significant growth inhibition (P < 0.05) was obtained for both doses and by both routes. These data provide further support for the view that retinoids have value for the treatment of ovarian cancer.

Estrogen regulation of transforming growth factor-alpha in ovarian cancer.

Transforming growth factor alpha (TGFalpha) may be induced by estrogen in estrogen responsive systems and can contribute to the growth-modulatory effects of this hormone. To test whether TGFalpha contributes to estrogen-regulated growth in ovarian cancers, we have compared the effects of 17beta-estradiol (E2) and TGFalpha in a range of ovarian carcinoma cell lines. Addition of E2 to the estrogen receptor (ER)-positive cell lines (PE01, PE04 and PE01CDDP) produced a 2-4 fold increase in TGFalpha protein concentrations in media conditioned by the cells. Both E2 and TGFalpha stimulated the growth of the PE01 and PE04 lines and inhibited the growth of the PE01CDDP line. Furthermore, the E2-mediated growth effects could be reversed by an epidermal growth factor (EGF) receptor-targeted antibody. E2 also down-regulated EGF receptor expression in ER-positive cell lines. In a series of primary ovarian tumors, higher concentrations of ER were associated with an increased percentage of tumors expressing TGFalpha mRNA and a decreased percentage expressing EGF receptor protein. All these data are consistent with E2 increasing production of TGFalpha in ER-positive ovarian cancer and this in turn acting through the EGF receptor to modulate growth in an autocrine manner.

Expression of the heat shock protein HSP27 in human ovarian cancer.

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.

Growth-control of human ovarian-carcinoma cells by insulin-like growth-factors.

The role of the insulin-like growth factors (IGFs) in 3 cultured human ovarian cancer cell lines (PEO1, PEO4, PEO14) was investigated. All three cell lines express mRNA for IGF-I and the PEO14 cell line expresses mRNA for IGF-II. Protein expression of IGF-II was demonstrated in the PEO14 and PEO4 cell lines. All 3 cell lines expressed mRNA for the IGF type I, IGF type II and insulin receptors; the presence of type I IGF receptors was confirmed by immuno-cytochemistry. IGF-I and insulin markedly stimulated the proliferation of PEO1 and PEO4 but not PEO14 cells while all 3 lines were insensitive to the addition of exogenous IGF-II.

Transforming growth-factor-Beta (tgf-Beta) messenger-RNA expression and growth-control in human lung squamous carcinoma cell-lines.

The pattern of TGF-beta mRNA expression and response to TGF-beta isoforms has been examined in three lung squamous carcinoma cell lines (NX002, CX140 and CX143). Expression for TGF-beta1 and TGF-beta2 but not TGF-beta3 was found in all 3 lines. TGF-beta1 and TGF-beta3 (but not TGF-beta2) inhibited the growth of the NX002 cell line in culture. The effect of TGF-beta1 on growth was accompanied with changes in the cell cycle. These data indicate that the potential for autocrine regulation by TGF-beta is present in lung squamous carcinoma cells.

12-O-tetradecanoylphorbol 13-acetate induced differentiation in human lung squamous carcinoma cells.

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.

Growth control by epidermal growth factor and transforming growth factor-alpha in human lung squamous carcinoma cells.

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Transforming growth factor-beta mRNA expression and growth control of human ovarian carcinoma cells.

The pattern of TGF beta expression and in vitro response to TGF beta has been defined in three ovarian carcinoma cell lines (PEO1, PEO4 and PEO14). Marked differences in both mRNA expression and growth responses were detected between the cell lines. All expressed mRNA for TGF beta 3, PEO1 and PEO4 but not PEO14 expressed mRNA for TGF beta 1, whereas PEO14 but not PEO1 and PEO4 expressed TGF beta 2. Growth of PEO14 cells in culture was markedly inhibited by both TGF beta 1 and beta 2. PEO1 cells were inhibited by TGF beta 1, but not TGF beta 2 whilst growth of PEO4 cells were not affected by exposure to either of these peptides. These data indicate that several elements of potential autocrine loops involving TGF beta's are present within ovarian cancer cells.

Characterisation and properties of a small cell lung cancer cell line and xenograft WX322 with marked sensitivity to alpha-interferon.

Controversy exists as to whether interferons usefully influence the growth of epithelial carcinomas. A small cell lung carcinoma (SCLC) cell line, WX322, has been derived which is greater than 1000-fold more sensitive to alpha-interferon (IFN) when grown in agar than other reported SCLC cell lines. The WX322 line has been characterised to prove its epithelial origin and its chemosensitivity compared with that of the NCI-H69 small cell line. The WX322 cell line expresses neuroendocrine and epithelial markers and possesses a morphology consistent with SCLC origin. A concentration of 5 IU ml-1 of IFN produced 50% inhibition of colony formation in agar in the WX322 line, whereas a concentration of greater than 10(5) IU ml-1 was required to produce a comparable effect with the NCI-H69 cell line. In contrast, WX322, possessed similar sensitivity to NCI-H69 cells when exposed to a range of cytotoxic agents. Analysis of the cell cycle indicated that IFN increased the percentage of cells in the G0/G1 phase for the WX322 cell line but increased the percentage in S phase for the NCI-H69 line. Growth of the xenograft, from which the cell line was derived, was also inhibited by IFN at doses greater than 10(5) IU/mouse/day. The WX322 cell line whether grown in agar or as a xenograft shows an unusually high sensitivity to IFN and provides an interesting model for studying mechanisms of IFN cytotoxicity to epithelial cells.