Human Ovarian Tumor Cell Lines Research Articles

Orphan Nuclear Receptor Nur77 Regulates Androgen Receptor Gene Expression in Mouse Ovary

The androgen receptor (AR) is a nuclear receptor that is expressed in growing follicles and involved in folliculogenesis and follicle growth. The orphan nuclear receptor, Nur77, also has an important role in steroid signaling and follicle maturation. We hypothesized that AR levels and androgen signaling through AR are regulated by Nur77 in the ovary. In the ovaries of Nur77 knockout mice (n = 5), real-time PCR results showed that the mRNA levels of AR and an androgen signaling target gene, Kitl, were decreased by 35% and 24%, respectively, relative to wild-type mice (n = 5), which suggested transcriptional regulation of AR by Nur77 in vivo. In cultured mouse granulosa cells and a steroidogenic human ovarian granulosa-like tumor cell line, KGN, mRNA and protein expression levels of AR were increased by overexpressing Nur77 but decreased by knocking down endogenous Nur77. Consistent with increased AR expression, chromatin immunoprecipitation showed that Nur77 bound to the NGFI-B response element (NBRE) in the AR promoter sequence. AR promoter activity was stimulated by Nur77 in HEK293T cells and attenuated in Nur77 knockout mouse granulosa cells (luciferase assay). Overexpression of Nur77 enhanced the androgenic induction of Kitl (200 nM; 48h), while knockout of Nur77 attenuated this induction. These results demonstrate that AR is regulated by Nur77 in the ovaries, and they suggest that the participation of Nur77 in androgen signaling may be essential for normal follicular development.

Functional Characterization of CLPTM1L as a Lung Cancer Risk Candidate Gene in the 5p15.33 Locus

Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

Chemerin inhibits IGF-1-induced progesterone and estradiol secretion in human granulosa cells

Chemerin is a novel adipokine involved in the regulation of adipocyte development, inflammation and metabolic functions. To date, no role of this adipokine in reproductive functions has been described. In the present study, we identified chemerin and its receptor, CMKLR1 (chemokine-like receptor 1), in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also investigated the effects of recombinant human chemerin (rhChem) on steroid production and on various signalling pathways. By RT-PCR immunoblotting and immunohistochemistry, we showed that chemerin and CMKLR1 are expressed in hGCs and KGN cells. By ELISA, we also found chemerin in human follicular fluid and we observed that in 8 of 10 women the chemerin level was at least 2-fold higher in follicular fluid than in plasma. rhChem (10 or 100 ng/ml) significantly decreased insulin-like growth factor-1 (IGF-1) (10(-8) M)-induced secretion of progesterone and estradiol (as determined by radioimmunoassay) but did not affect basal-or FSH (10(-8) M)-induced steroid secretion in hGCs and KGN cells. In parallel, it also decreased IGF-1-induced p450 aromatase protein levels without affecting the protein levels of other factors involved in steroidogenesis (steroidogenic acute regulatory protein, 3-beta-hydroxysteroid dehydrogenase and p450 side-chain cleavage enzyme) in hGCs cells. All these changes were associated with a decrease in the IGF-1-induced tyrosine phosphorylation of IGF-1 receptor beta subunit and phosphorylation of mitogen-activated protein kinase extracellular signal-regulated kinases 1/2 (MAPK ERK1/2) and Akt. In hGCs and KGN cells, rhChem also decreased IGF-1-induced thymidine incorporation. Finally, we showed that rhChem rapidly activates MAPK ERK1/2, MAPK P38 and Akt phosphorylation and more slowly AMP-activated protein kinase phosphorylation under basal conditions (no IGF-1 or FSH) in primary hGC cells. Taken together, chemerin and its receptor (CMKLR1) are present and active in hGCs. Chemerin reduces IGF-1-induced steroidogenesis and cell proliferation through a decrease in the activation of IGF-1R signalling pathways in primary hGCs.

Effect of RNA Interference of BID and BAX mRNAs on Apoptosis in Granulosa Cell-derived KGN Cells

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

In vitro screening of ovarian tumor specific peptides from a phage display peptide library

To develop more biomarkers for diagnosis and therapy of ovarian cancer, a 12-mer phage display library was used to isolate peptides that bound specifically to the human ovarian tumor cell line SK-OV-3. After five rounds of in vitro screening, the recovery rate of phages showed a 69-fold increase over the first round of washings and a group of phage clones capable of binding to SK-OV-3 cells were obtained. A phage clone named Z1 with high affinity and specificity to SK-OV-3 cells was identified in vitro. More importantly, the synthetic biotin-labeled peptide, ZP1 (=SVSVGMKPSPRP), which corresponded to the sequence of the inserted fragment of Z1, demonstrated a high specificity to SK-OV-3 cells especially when compared to other cell lines (A2780 and 3T3). ZP1 might therefore be a biomarker for targeting drug delivery in ovarian cancer therapy.

Effect of FSH and its Glycosylation Variants on Proliferation, Steroid and Inhibin Production in a Human Ovarian Granulosa-Like Tumor Cell Line (KGN).

Effect of FSH and its Glycosylation Variants on Proliferation, Steroid and Inhibin Production in a Human Ovarian Granulosa-Like Tumor Cell Line (KGN).

Abstract 4412: In vitro activity of the novel tubulin active agent BAL27862 in MDR1(+) and MDR1(−) human breast and ovarian cancer variants selected for resistance to taxanes

Abstract BAL27862 is a synthetic small molecule that induces apoptosis due to the inhibition of tubulin polymerization via a potentially new mechanism of action. The activity of this novel compound was evaluated in human breast and ovarian cancer cell line variants selected for resistance by continuous exposure to taxanes. BAL27862 retained activity against MDR1 (ABCB1)/P-glycoprotein-mediated resistance variants, unlike vinca alkaloids and taxanes. Activity was also observed in the taxane-resistant ovarian 1A9/TxTP50 tumor cell line, which overexpresses TUBB3 and was approximately 35x resistant to taxanes. Moreover, BAL27862 retained activity in ovarian cancer cells with acquired mutations in the class I (M40) β-tubulin isotype (1A9/PTX10, Phe270->Val; 1A9/PTX22, Ala364->Thr) which confer taxane resistance. The MCF-7/TxTP50 breast tumor variant was 9x resistant to taxanes, and 5x resistant to epothilone B, yet retained sensitivity to BAL27862, the vinca alkaloids (vinblastine and vincristine), colchicine and anthracyclines. These cells have significantly reduced levels of the taxane-binding target, the class I β-tubulin isotype, and reduced levels of total α- and β-tubulin relative to wild-type cells, alterations which may be responsible for a 34% reduction in bound BODIPY-paclitaxel as detected by FACS analysis. Evidence of resistance to BAL27862 (3x) was only detected in the ovarian OVCAR-3/TxTP5 variant, which was also resistant to taxanes (8x), the vinca alkaloids (4x) and colchicine (2x) and ixabepilone (2x). Finally, silencing TUBB3 using a specific siRNA sensitized cells to taxanes but did not affect BAL27862 activity in both wild-type MCF-7 and OVCAR-3 cells relative to appropriate controls, suggesting that TUBB3 status may not influence BAL27862 response. Thus, BAL27862 is a new agent with a distinct in vitro activity profile against a broad panel of human breast and ovarian tumor cell lines resistant to standard tubulin-interacting agents. These data strongly support further development of BAL27862 as a novel anticancer agent. 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 4412.

89Zr-trastuzumab PET visualises HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a human tumour xenograft

NVP-AUY922, a potent heat shock protein (HSP) 90 inhibitor, downregulates the expression of many oncogenic proteins, including the human epidermal growth factor receptor-2 (HER2). Because HER2 downregulation is a potential biomarker for early response to HSP90-targeted therapies, we used the 89Zr-labelled HER2 antibody trastuzumab to quantify the alterations in HER2 expression after NVP-AUY922 treatment with HER2 positron emission tomography (PET) imaging. The HER2 overexpressing human SKOV-3 ovarian tumour cell line was used for in vitro experiments and as xenograft model in nude athymic mice. In vitro HER2 membrane expression was assessed by flow cytometry and a radio-immuno assay with 89Zr-trastuzumab. For in vivo evaluation, mice received 50 mg/kg NVP-AUY922 intraperitoneally every other day. 89Zr-trastuzumab was injected intravenously 6 d before NVP-AUY922 treatment and after 3 NVP-AUY922 doses. MicroPET imaging was performed at 24, 72 and 144 h post tracer injection followed by ex-vivo biodistribution and immunohistochemical staining. After 24 h NVP-AUY922 treatment HER2 membrane expression showed profound reduction with flow cytometry (80%) and radio-immuno assay (75%). PET tumour quantification, showed a mean reduction of 41% ( p = 0.0001) in 89Zr-trastuzumab uptake at 144 h post tracer injection after NVP-AUY922 treatment. PET results were confirmed by ex-vivo 89Zr-trastuzumab biodistribution and HER2 immunohistochemical staining. NVP-AUY922 effectively downregulates HER2, which can be monitored and quantified in vivo non-invasively with 89Zr-trastuzumab PET. This technique is currently under clinical evaluation and might serve as an early biomarker for HSP90 inhibition in HER2 positive metastatic breast cancer patients.

Abstract C109: NOV-002 suppresses tumor cell growth by modulating redox-sensitive cell signaling

Abstract Regulation of cell proliferation/survival is complex, impacted by multiple endogenous and exogenous stimuli capable of triggering overlapping and competing signaling pathways whose net effects can be quite cell dependent. Changes in redox status is one regulator of cell life/death decisions under both physiologic and pathologic conditions. Here we report on the anti-proliferative effects of a pharmacologically generated oxidative signal in human tumor cells using NOV-002, a glutathione disulfide-mimetic in advanced clinical trials for oncological indications. NOV-002 has demonstrated efficacy (increased survival and/or decreased tumor growth) in non-small cell lung, breast and ovarian cancers when combined with standard chemotherapeutic agents. In addition, treatment with NOV-002 mitigates chemotherapy-associated hematological toxicity. Pre-clinical data has linked this effect to proliferation of bone marrow progenitor cells subsequent to generation of an oxidative signal, intracellularly and at the cell surface of myeloid lineage cells, leading to activation of multiple kinases known to regulate cell proliferation (e.g. MAP kinases, JAK/STAT kinases). The data presented here extend redox modulation studies with NOV-002 to tumor cells. SKOV3 cells (a human ovarian tumor cell line) were exposed to NOV-002 (250 uM). Within 5 min after a single treatment, cellular levels of reactive oxygen species (ROS) were significantly elevated indicating the generation of an oxidative signal by NOV-002. This was not, however, sufficient to influence redox-sensitive cell signaling or tumor cell proliferation rate. In contrast, more prolonged exposure to NOV-002 (daily treatment for 5 days) resulted in a sustained elevation in ROS and dose-dependent activation of the proliferation-regulating MAP kinase, JNK, as evidenced by an increase in its active, phosphorylated form. Most importantly, this redox-activated cell signaling effect was associated with a significantly decreased proliferation rate of the SKOV3 cells that persisted even after stopping treatment with NOV-002. Thus, oxidative signal generation and MAP kinase pathway activation by NOV-002 appears to result in cell-type dependent effects on proliferation/survival. In myeloid lineage/bone marrow cells, chronic exposure to NOV-002 leads to increased proliferation while in tumor cells the result is a decrease in proliferation. These dichotomous effects may contribute to the unique clinical profile that NOV-002 has demonstrated to date — increased anti-tumor efficacy and survival combined with enhanced recovery from chemotherapy-induced hematological toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C109.

Synthesis, Characterization, and In Vitro Antitumor Activity of New Amidineplatinum(II) Complexes Obtained by Addition of Ammonia to Coordinated Acetonitrile

AbstractNew cis‐ and trans‐dichloridoplatinum(II) complexes, which contain two amidine ligands or one amidine and one ammine ligand, cis‐ and trans‐[PtCl2{(Z)‐HN=C(NH2)CH3}2] (1) and cis‐ and trans‐[PtCl2(NH3){(Z)‐HN=C(NH2)CH3}] (2), have been prepared from the corresponding nitrile complexes by amminolysis in thf solution. All synthesized compounds were characterized by elemental analysis, ESI‐MS, and IR and NMR spectroscopy. Amidines are isosters of iminoethers and ketimines. The trans isomers of the platinum complexes of the latter are endowed with an unexpectedly high antitumor activity. An important feature of complexes 1 and 2, as compared to iminoethers, is the exclusive preference for the Z configuration of the amidine ligand(s). The tumor cell growth inhibitory potency of the amidine compounds was tested towards a pair of human ovarian tumor cell lines A2780 (cancer cells sensitive to cisplatin) and A2780cisR (cancer cells with acquired resistance to cisplatin), and compared to that of cisplatin. From the obtained results it appears that the resistance factor is lower for cis‐amidine complexes as compared to cisplatin, and for trans compounds it is lower as compared to cis compounds.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross‐resistance to a wide range of DNA‐damaging drugs

Although DNA-damaging agents are among the most effective anticancer drugs in clinical use, their overall effectiveness is limited by the development of cross-resistance to these drugs. Given that histone deacetylase (HDAC) inhibitors increase the acetylation of core histones, resulting in an open chromatin configuration that is more accessible to DNA-targeting agents, we examined whether HDAC inhibitors might enhance the cytotoxicity of DNA-damaging drugs in six human ovarian tumor cell lines that exhibit different cisplatin sensitivities. Low concentrations of HDAC inhibitors, which alone exhibited little cytotoxicity, markedly enhanced the induction of apoptotic cell death not only by cisplatin but also by a wide variety of DNA-targeting anticancer drugs in these tumor cell lines, irrespective of their sensitivities to the respective drugs. In contrast, HDAC inhibitors did not increase the cytotoxicity of metabolic antagonists or microtubule-targeting agents. HDAC inhibitors potentiated both the phosphorylation of histone H2AX on serine-139 (a marker of DNA double-strand breaks) as well as the accumulation of reactive oxygen species induced by DNA-damaging agents in tumor cells. The enhanced generation of reactive oxygen species appeared to be responsible for the enhanced apoptotic cell death induced by the combination of these drugs. These results indicate that the combination of an HDAC inhibitor with a wide variety of DNA-damaging agents is a promising chemotherapeutic strategy for the eradication of tumor cells, regardless of whether the cells are sensitive or resistant to the DNA-damaging anticancer drugs.

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.

Dihydrodiol dehydrogenases regulate the generation of reactive oxygen species and the development of cisplatin resistance in human ovarian carcinoma cells

We have previously demonstrated that overexpression of dihydrodiol dehydrogenase isoform 1 (DDH1) or DDH2 leads to the induction of drug resistance to platinum based drugs in human ovarian, lung, cervical and germ cell tumor cell lines. DDH belongs to a family of aldoketo reductases that are involved in the detoxification of several endogenous and exogenous substrates. DDH1 and DDH2 in particular have been shown to be involved in the detoxification (activation?) of polycyclic aromatic hydrocarbons (PAH). Based on the involvement of DDH in the detoxification of electrophilic PAH intermediates, the effect of DDH on the production of reactive oxygen species (ROS) in a cisplatin-sensitive and -resistant human ovarian carcinoma cell line was investigated in the current study. In addition to the overexpression of DDH1 and DDH2, increased expression of DDH3 was demonstrated in the cisplatin-resistant 2008/C13* cells, compared to the parental 2008 cells. However, as assessed by RT-PCR, neither cell line expressed DDH4. The 2008/C13* cells were eightfold resistant to cisplatin, and transfection experiments utilizing cisplatin-sensitive 2008 cells suggest that this could be mediated by overexpression of either DDH1, DDH2, or DDH3. The 2008/C13* cells had lower basal intracellular ROS level as compared to the 2008 cells and ROS production was decreased in the recombinant 2008 cells with forced, constitutive overexpression of either, DDH1, DDH2, or DDH3. Transfection of siRNA against DDH1 or DDH2 in the cisplatin-resistant 2008/C13* cells not only significantly decreased their cisplatin-resistance index (as assayed by MTT and colony formation assay) but also led to an increase in the basal levels of ROS production (although transfection of siRNA against DDH3 resulted in cell death). The 2008/C13* cells were found to be cross-resistant to the cytotoxic effects of hydrogen peroxide and tert-butyl hydroperoxide and knockdown of either DDH1 or DDH2 expression (using siRNA) resulted in sensitization of the resistant cells to these agents. These results support the conclusion that the increased levels of DDH in the 2008/C13* cells are directly responsible for the reduced production of ROS and that this may play a role in the development of cisplatin resistance.

Anti‐apoptotic activity of porcine cFLIP in ovarian granulosa cell lines

In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIP(S) and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia.

Human ovarian tumour-derived chaperone-rich cell lysate (CRCL) elicits T cell responses in vitro

Tumour-derived chaperone-rich cell lysate (CRCL), which is made up of numerous heat shock proteins, has been used successfully to generate tumour-specific T cell responses and protective immunity against a wide range of murine tumours. In this study, we have investigated the potency of human ovarian cancer-derived CRCL to activate dendritic cells (DC) and to generate tumour-specific T cells in vitro. CRCL was generated from primary ovarian cancers and SKOV3-A2, a HER2/neu, Wilm's tumour gene 1 (WT1) and human leucocyte antigen (HLA)-A2 positive human ovarian tumour cell line. Peripheral blood mononuclear cells from both HLA-A2(+) healthy donors and HLA-A2(+) ovarian cancer patients were stimulated weekly with autologous DC loaded with ovarian tumour-derived CRCL. After four to six stimulations in vitro, specific cytokine secretion and cytotoxicity were measured. CRCL promoted interleukin (IL)-12 secretion and enhanced the immunostimulatory capacity of DC. T cells from healthy controls and from ovarian cancer patients secreted higher amounts of interferon-gamma following in vitro restimulation with ovarian cancer-derived CRCL than with HER2/neu or WT1 peptide-pulsed DC. We were also able to generate cytotoxic T lymphocyte activity against cancer-specific antigens such as HER2/neu and WT1 from all healthy donors, but from only one of the four ovarian cancer patients with bulky disease. These preliminary results substantiate further the concept that CRCL may prove to be a potent adjuvant for women suffering from ovarian cancer and that this personalized vaccine may be a promising approach for active immunotherapy.

185 Genetic analysis of radioresistance in a human ovarian tumor cell line

185 Genetic analysis of radioresistance in a human ovarian tumor cell line

1470 POSTER Reduction and activation of RH1 by NADPH cytochrome P450 reductase

Purpose: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background.Methods: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol.Results: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to ∼200 nmol/min/mg, and the NQO1 activity in KHT/iv increased ∼10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis.Conclusions: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.

1472 POSTER A phase I study with fosfluridine tidoxil, a novel oral fluoropyrimidine, in patients with advanced solid tumors

Purpose: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background.Methods: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol.Results: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to ∼200 nmol/min/mg, and the NQO1 activity in KHT/iv increased ∼10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis.Conclusions: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.

A phase I pharmacokinetic and pharmacodynamic study of BGC9331 and carboplatin in relapsed gynaecological malignancies

BGC9331 is a rationally designed, specific nonpolyglutamatable thymidylate synthase (TS) inhibitor that is active in gynaecological malignancies. In the light of the sensitivity of human ovarian tumour cell lines to BGC9331 and non-cross resistance to platinum drugs, we studied the combination BGC9331/carboplatin (BCA) in a phase I (PI) pharmacokinetic (PK) and pharmacodynamic (PD) study in platinum pretreated gynaecological malignancies. Patients were ⩾18 years or over, with a histologically confirmed gynaecological malignancy, radiological evidence of relapse, and a platinum treatment free interval of at least 6 months. Up to three prior lines of chemotherapy were permitted. Carboplatin (AUC5) and BGC9331 were administered on day 1, and BGC9331 was also given on day 8 of a 21-day cycle. In total, 14 patients were enrolled, and treated with BGC9331 at four dose levels, 40, 65, 85 and 100 mg m−2. The principal grade 3 and 4 haematological toxicity was neutropaenia. The principal nonhaematological toxicities were lethargy and nausea. Dose-limiting toxicities were seen in two patients at 100 mg m−2 BGC9331 (grade 4 neutropaenia >7 days, and grade 4 fatigue >7 days). Plasma BGC9331 was measured by an ELISA that was adapted for use in humans. Carboplatin was assayed by flameless atomic absorption spectrometry. There was no PK interaction between the two drugs. Plasma deoxyuridine was elevated indicating TS inhibition to at least day 12. Antitumour activity was observed in four out of 14 (28%) of patients. In conclusion, the combination of BGC9331 and carboplatin is well tolerated with no significant PK interaction between the two drugs. There is evidence of TS inhibition with the combination. We have demonstrated antitumour activity in platinum pretreated gynaecological malignancy. Further exploration of this combination in this disease is warranted.

Pooled peptides from HER-2/neu-overexpressing primary ovarian tumours induce CTL with potent antitumour responses in vitro and in vivo

Unfractionated peptides (MW: up to 10 kDa), derived from HLA-A2.1 positive (+) HER-2/neu-overexpressing primary tumour cell acid cell extracts (ACE), were successfully used to generate in vitro cytotoxic T lymphocytes (CTL). Primary tumour cells were collected from peritoneal malignant effusions of patients with ovarian cancer. Acid cell extracts-induced CTL specifically lysed in an HLA-A2-restricted manner HER-2/neu+ autologous primary tumour cells as well as HER-2/neu+ tumour cell lines. In addition, adoptive transfer of such CTL significantly prolonged the survival of SCID mice xenografted with HLA-A2.1+, HER-2/neu+ human breast and ovarian tumour cell lines. Acid cell extracts collected from HLA-A2.1+ HER-2/neu negative (−) primary ovarian tumours induced HLA-A2.1-restricted CTL with weak in vitro and in vivo antitumour capacity, suggesting that HER-2/neu peptides within ACE from HER-2/neu-overexpressing primary ovarian tumour cells are immunodominant. The results presented herein serve as a rationale for the initiation of vaccination studies in patients with HER-2/neu-overexpressing ovarian tumours utilising autologous tumour-derived ACE.