A novel function of HER2/Neu in the activation of G2/M checkpoint in response to γ-irradiation.

In response to γ-irradiation (IR)-induced DNA damage, activation of cell cycle checkpoints results in cell cycle arrest, allowing time for DNA repair before cell cycle re-entry. Human cells contain G1 and G2 cell cycle checkpoints. While G1 checkpoint is defective in most cancer cells, commonly due to mutations and/or alterations in the key regulators of G1 checkpoint (for example, p53, cyclin D), G2 checkpoint is rarely impaired in cancer cells, which is important for cancer cell survival. G2 checkpoint activation involves activation of ataxia telangiectasia-mutated (ATM)/ATM- and rad3-related (ATR) signalings, which leads to the inhibition of Cdc2 kinase and subsequent G2/M cell cycle arrest. Previous studies from our laboratory show that G2 checkpoint activation following IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. As HER receptor tyrosine kinases (RTKs), which have important roles in cell proliferation and survival, have been shown to activate ERK1/2 signaling in response to various stimuli, we investigated the role of HER RTKs in IR-induced G2/M checkpoint response in breast cancer cells. Results of the present studies indicate that IR exposure resulted in a striking increase in the phosphorylation of HER1, HER2, HER3 and HER4 in MCF-7 cells, indicative of activation of these proteins. Furthermore, specific inhibition of HER2 using an inhibitor, short hairpin RNA and dominant-negative mutant HER2 abolished IR-induced activation of ATM/ATR signaling, phosphorylation of Cdc2-Y15 and subsequent induction of G2/M arrest. Moreover, the inhibition of HER2 also abrogated IR-induced ERK1/2 phosphorylation. In contrast, inhibition of HER1 using specific inhibitors or decreasing expression of HER3 or HER4 using short hairpin RNAs did not block the induction of G2/M arrest following IR. These results suggest an important role of HER2 in the activation of G2/M checkpoint response following IR.

Protein phosphatase 2A has an essential role in the activation of gamma-irradiation-induced G2/M checkpoint response.

G2/M checkpoint activation after DNA damage results in G2/M cell cycle arrest that allows time for DNA repair before the entry of cells into mitosis. Activation of G2/M checkpoint involves a series of signaling events, which include activation of ataxia telangiectecia-mutated and Rad3-related (ATR) and Chk1 kinases and inhibition of Cdc2/Cyclin B activity. Studies presented in this report show that serine (Ser)/threonine (Thr) protein phosphatase 2A (PP2A) has an important role in G2/M checkpoint activation in response to gamma-irradiation (IR) exposure. Using PP2A inhibitors, as well as siRNA targeting various forms of Ser/Thr protein phosphatases, results presented in this report show that specific PP2A inhibition abrogates IR-induced activation of ATR and Chk1 kinases, as well as phosphorylation of Cdc2-Tyr15, and attenuates IR-induced G2/M arrest. These results suggest an important regulation of PP2A on IR-induced G2/M checkpoint signaling response.

Irradiation-induced G2/M checkpoint response requires ERK1/2 activation.

Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.

Thromboembolism following removal of femoral venous apheresis catheters in patients with breast cancer.

BACKGROUND: Apheresis catheters have simplified collection of peripheral blood stem cells (PBSC), but may be associated with thrombosis of the instrumented vessels. We performed a retrospective analysis to study the prevalence of thromboembolism associated with the use of femoral apheresis catheters in patients with breast cancer. PATIENTS AND METHODS: Patients were participants in clinical trials of high-dose chemotherapy with autologous PBSC rescue. They underwent mobilization with either high-dose cyclophosphamide (n = 21) or cyclophosphamide/paclitaxel (n = 64), followed by filgrastim. Double lumen catheters (12 or 13 Fr) were placed in the femoral vein and removed within 12 h of the last apheresis procedure. Apheresis was performed using a continuous flow cell separator and ACD-A anticoagulant. Thromboembolism was diagnosed by either venous ultrasonography or ventilation-perfusion scan. RESULTS: Nine of 85 patients (10.6%) undergoing large volume apheresis with use of a femoral catheter developed thromboembolic complications. Pulmonary embolus (PE) was diagnosed in five and femoral vein thrombosis in four patients. Four of the five patients who developed PE were symptomatic; one asymptomatic patient had a pleural-based, wedge-shaped lesion detected on a staging computed tomography scan. The mean number of apheresis procedures was 2.4 (range one to four) and the mean interval between removal of the apheresis catheter and diagnosis of thrombosis was 17.6 days. In contrast, none of 18 patients undergoing apheresis using jugular venous access and none of 54 healthy allogeneic donors undergoing concurrent filgrastim-mobilized PBSC donation (mean 1.7 procedures/donor) using femoral access experienced thromboembolic complications. CONCLUSIONS: Thromboembolism following femoral venous catheter placement for PBSC collection in patients with breast cancer may be more common than previously recognized. Healthy PBSC donors are not at the same risk. Onset of symptoms related to thrombosis tended to occur several weeks after catheter removal. This suggests that the physicians not only need to be vigilant during the period of apheresis, but also need to observe patients for thromboembolic complications after the catheter is removed. The long interval between the removal of apheresis catheter and the development of thromboembolism may have a potential impact on prophylactic strategies developed in future, such as the duration of prophylactic anticoagulation. Avoidance of the femoral site in breast cancer patients, and close prospective monitoring after catheter removal, are indicated.

Phase I clinical trial of alitretinoin and tamoxifen in breast cancer patients: toxicity, pharmacokinetic, and biomarker evaluations.

PURPOSE: To determine the overall and dose-limiting toxicities (DLTs) of alitretinoin (9-cis-retinoic acid) in combination with tamoxifen and the pharmacokinetics of alitretinoin alone and when combined with tamoxifen in patients with metastatic breast cancer. The effect of tamoxifen and alitretinoin on MIB-1, a marker of proliferation, in unaffected breast tissue was explored. PATIENTS AND METHODS: Eligible patients had metastatic breast cancer. Previous tamoxifen therapy was allowed. Planned dose levels for alitretinoin ranged from 50 to 140 mg/m2/d with 20 mg/d tamoxifen in all patients after 4 weeks of alitretinoin as a single agent. Plasma concentrations of alitretinoin and retinol were measured at baseline and after 1, 2, and 3 months. Breast core biopsies were obtained at baseline and after 2 months of therapy. RESULTS: Twelve patients with metastatic breast cancer received a total of 86 cycles of therapy. At 90 mg/m2/d, three of five patients experienced a DLT: grade 3 headache, grade 3 hypercalcemia, and grade 3 noncardiogenic pulmonary edema. At 70 mg/m2/d, one of six patients experienced a DLT (headache), and this level was considered the maximal tolerated dose in this study. Three toxicities occurred that had not been reported previously with alitretinoin: an asymptomatic delay in dark adaptation, a marked decrease in high-density lipoprotein cholesterol, and the occurrence of enthesopathy. Two of the nine assessable patients had a durable clinical response: one partial response and stable disease for 18 months and one complete response in continuous remission for 48+ months. Both responding patients were estrogen receptor-positive and had had previous tamoxifen therapy. There was a high degree of interpatient variability of plasma alitretinoin concentrations, although a significant decline in alitretinoin plasma levels over time was observed. MIB-1 scores declined in four of the eight paired breast specimens obtained. CONCLUSION: The combination of tamoxifen and alitretinoin is well tolerated and has antitumor activity in metastatic breast cancer. The recommended phase II dose is 70 mg/m2/d with 20 mg/d tamoxifen.

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter.

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 microg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 microg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein.

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.

Detection of circulating tumor cells and micrometastases in stage II, III, and IV breast cancer patients utilizing cytology and immunocytochemistry.

Evaluation for circulating tumor cells and bone marrow micrometastases has generated considerable interest due to a potential association with disease recurrence and poor prognosis. In this study, we examined bone marrow and apheresis samples from Stage II, III, and IV patients (n 120) enrolled in various clinical breast cancer trials at the National Institutes of Health/National Cancer Institute. For each patient sample, two Diff-Quik-stained cytospins were reviewed for morphology, and approximately 1 x 10(6) cells were analyzed for the expression of cytokeratins using an avidin-biotin immunoperoxidase method. Keratin-positive malignant cells appearing as single cells or in small clusters were detected in bone marrow samples from Stage IV patients only (9/68, 13%) and detected in apheresis samples from both Stage III and IV patients (13/245, 5%). These findings indicate that the combination of cytomorphology with immunocytochemistry can be utilized for the investigation of circulating tumor cells and bone marrow micrometastases, and that positive results appear to correlate with high tumor stage/burden.

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12).

IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.

Phase I and pharmacokinetic study of farnesyl protein transferase inhibitor R115777 in advanced cancer.

PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetic profile of the farnesyl protein transferase inhibitor R115777 when administered orally bid for 5 days every 2 weeks. PATIENTS AND METHODS: Twenty-seven patients with a median age of 58 years received 85 cycles of R115777 using an intrapatient and interpatient dose escalation schema. Drug was administered orally at escalating doses as a solution (25 to 850 mg bid) or as pellet capsules (500 to 1300 mg bid). Pharmacokinetics were assessed after the first dose and the last dose administered during cycle 1. RESULTS: Dose-limiting toxicity of grade 3 neuropathy was observed in one patient and grade 2 fatigue (decrease in two performance status levels) was seen in four of six patients treated with 1,300 mg bid. The most frequent clinical grade 2 or 3 adverse events in any cycle included nausea, vomiting, headache, fatigue, anemia, and hypotension. Myelosuppression was mild and infrequent. Peak plasma concentrations of R115777 were achieved within 0.5 to 4 hours after oral drug administration. The elimination of R115777 from plasma was biphasic, with sequential half-lives of about 5 hours and 16 hours. There was little drug accumulation after bid dosing, and steady-state concentrations were achieved within 2 to 3 days. The pharmacokinetics were dose proportional in the 25 to 325 mg/dose range for the oral solution. Urinary excretion of unchanged R115777 was less than 0.1% of the oral dose. One patient with metastatic colon cancer treated at the 500-mg bid dose had a 46% decrease in carcinoembryonic antigen levels, improvement in cough, and radiographically stable disease for 5 months. CONCLUSION: R115777 is bioavailable after oral administration and has an acceptable toxicity profile. Based upon pharmacokinetic data, the recommended dose for phase II trials is 500 mg orally bid (total daily dose, 1, 000 mg) for 5 consecutive days followed by 9 days of rest. Studies of continuous dosing and studies of R115777 in combination with chemotherapy are ongoing.

BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G(1) or G(2) phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer.

PURPOSE: N-(4-hydroxyphenyl) retinamide (¿4-HPR, Fenretinide; R.W. Johnson Pharmaceutical Research Institute, Springhouse, PA) and tamoxifen (TAM) have synergistic antitumor and chemopreventive activity against mammary cancer in preclinical studies. We performed a pilot study of this combination in women at high risk for developing breast cancer. PATIENTS AND METHODS: Thirty-two women were treated with four cycles of 4-HPR, 200 mg orally (PO) for 25 days of each 28-day cycle, and TAM, 20 mg PO once daily for 23 months beginning after 1 month of 4-HPR alone. Tolerability, dark adaptometry, tissue biopsies, and retinoid plasma concentrations (Cp) were evaluated. RESULTS: Symptomatic reversible nyctalopia developed in two patients (6%) on 4-HPR, but 16 (73%) of 22 patients had reversible changes in dark adaptation, which correlated with relative decrease in Cp retinol (P

BRCA1 signals ARF-dependent stabilization and coactivation of p53.

The hereditary breast and ovarian tumor suppressor BRCA1 can activate p53-dependent gene expression. We show here that BRCA1 increases p53 protein levels through a post-transcriptional mechanism. BRCA1-stabilized p53 has increased sequence-specific DNA-binding and transcriptional activity. BRCA1 does not stabilize p53 in p14ARF-deficient cells. A deletion mutant of BRCA1 which inhibits p53-dependent transcription confers resistance to topoisomerase II-targeted chemotherapy. Our results suggest that BRCA1 may trigger the p53 pathway through two potentially separate mechanisms: accumulation of p53 through a direct or indirect induction of p14ARF as well as direct transcriptional coactivation of p53. BRCA1 may also enhance chemosensitivity and repair of DNA damage through binding to and coactivation of p53.

In vivo studies of adenovirus-mediated p53 gene therapy for cis-platinum-resistant human ovarian tumor xenografts.

We have recently reported that mutations of the tumor suppressor p53 gene are associated with the development of resistance to cis-platinum in human ovarian cancer cells, and that adenovirus-mediated reintroduction of the wild-type p53 (wtp53) gene in ovarian tumor cells resulted in the sensitization of tumor cells to cis-diamminedichloroplatinum (II) (CDDP). The purpose of this study was to evaluate whether i.p. treatment of CDDP-resistant tumor cells expressing mutant p53 (mutp53) with a recombinant adenovirus expressing wtp53 (Adwtp53) would result in the sensitization of resistant cells to CDDP. In order to determine whether i.p. injection of a recombinant adenovirus would result in expression of the transgene in tumor cells growing intraperitoneally, we first injected A2780/CP cells in nude mice and 10 days later the mice were injected i.p. with a recombinant adenovirus expressing beta-galactosidase (Ad beta-gal). Twenty-four hours following i.p. injection of Ad beta-gal, tumors were removed and stained for beta-gal. While tumors showed extensive staining for beta-gal, indicating internalization of adenovirus and the expression of the transgene in tumors, no expression of beta-gal protein was detected in liver. I.p. treatment of A2780/CP tumor xenografts with Adwtp53 caused extensive tumor cell death, which was further enhanced by CDDP. Treatment with Adwtp53 (5 x 10(7) pfu/day, 3-5 treatments) resulted in a significant decrease in tumor volume and increase in animal survival compared to either no treatment or treatment with vector alone without p53 gene. Additional therapy with CDDP (1 mg/kg/day x 3-4) further reduced tumor volume and increased survival (30-40%), suggesting that combination therapy of Adwtp53 and CDDP was better than single agents alone. Our results indicate that i.p. dosing with adenovirus-mediated wtp53 gene therapy could be beneficial in combination with CDDP for the treatment of ovarian tumors expressing mutp53.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

A novel adenoviral vector expressing human Fas/CD95/APO-1 enhances p53-mediated apoptosis.

Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.

Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1).

Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transport-mediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 non-coding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

Tamoxifen and fenretinide in women with metastatic breast cancer.

BACKGROUND: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factor-beta (TGF-beta) levels and serum lipids was also examined. PATIENTS AND METHODS: Thirty-one patients were treated with tamoxifen (20 mg p.o. daily), and fenretinide (400 mg p.o. daily with a 3-day drug holiday each month). Plasma TGF-beta testing was performed using isoform specific sandwich ELISA. RESULTS: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ER-negative patients, and three hormone therapy naive ER-positive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of -13.5 mg/dl; p = 0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18 mg/dl, p = 0.0001, a 35% increase) with tamoxifen and fenretinide therapy. TGF-beta1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated. CONCLUSIONS: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400 mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.