Endoplasmic reticulum/cytosolic localization of von Hippel-Lindau gene products is mediated by a 64-amino acid region.

Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes both the familial cancer syndrome VHL disease and corresponding sporadic tumor types, including renal-cell carcinoma. Subcellular localization of VHL gene products was determined by indirect immunofluorescence. Both native and exogenously expressed VHL proteins displayed a cytoplasmic peri-nuclear immunostaining pattern, which co-localized with markers for the endoplasmic reticulum (ER).

Novel combinations with oxaliplatin.

Oxaliplatin (Eloxatin) is a novel antineoplastic platinum derivative that may exert its cytotoxic effects by blocking DNA replication/transcription, thus resulting in cell death in proliferating cells, as well as apoptosis. Oxaliplatin is often more potent than other platinums such as cisplatin (Platinol) in vitro, and shows greater efficacy in preclinical studies against many tumor cell lines, including some that are resistant to cisplatin and carboplatin (Paraplatin). Oxaliplatin is approved for use with the fluoropyrimidines in the treatment of metastatic colorectal cancer in Asia, Latin America, and Europe.

VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling.

Mutations in the von Hippel-Lindau (VHL) gene are involved in the family cancer syndrome for which it is named and the development of sporadic renal cell cancer (RCC). Reintroduction of VHL into RCC cells lacking functional VHL [VHL(-)] can suppress their growth in nude mice, but not under standard tissue culture conditions. To examine the hypothesis that the tumor suppressor function of VHL requires signaling through contact with extracellular matrix (ECM), 786-O VHL(-) RCC cells and isogenic sublines stably expressing VHL gene products [VHL(+)] were grown on ECMs.

Cell-cycle dysregulation in breast cancer: breast cancer therapies targeting the cell cycle.

Breast cancer is the most commonly diagnosed cancer in American women. The underlying mechanisms that cause aberrant cell proliferation and tumor growth involve conserved pathways, which include components of the cell cycle machinery. Proto-oncogenes, growth factors, and steroids have been implicated in the pathogenesis of breast cancer.

Caveolins in cholesterol trafficking and signal transduction: implications for human disease.

Caveolins are a family of proteins that coat the cytoplasmic face of caveolae, vesicular invaginations of the plasma membrane. These proteins are central to the organization of the proteins and lipids that reside in caveolae. Caveolins transport cholesterol to and from caveolae, and they regulate the activity of signaling proteins that reside in caveolae.

Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells.

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP).

The mechanism of transport of the multitargeted antifolate (MTA) and its cross-resistance pattern in cells with markedly impaired transport of methotrexate.

MTA (LY231514) is an antifolate that targets multiple folate-dependent enzymes. In this report, MTA transport was characterized in wild-type L1210 cells and variants with impaired membrane transport or polyglutamation. MTA influx via the reduced folate carrier was somewhat faster (approximately 30%) than that for methotrexate (MTX).

Sensitivity to 5,10-dideazatetrahydrofolate is fully conserved in a murine leukemia cell line highly resistant to methotrexate due to impaired transport mediated by the reduced folate carrier.

A murine leukemia cell line was identified that is highly resistant to methotrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dideazatetrahydrofolate (DDATHF). A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in drug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell line markedly increased DDATHF influx (32x) but only modestly increased influx of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively).

Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL.

Phase II trial of doxorubicin and docetaxel plus granulocyte colony-stimulating factor in metastatic breast cancer: Eastern Cooperative Oncology Group Study E1196.

Purpose: The purpose of this multi-institutional phase II trial was to evaluate the efficacy and toxicity of doxorubicin and docetaxel plus granulocyte colony-stimulating factor (G-CSF) in patients with metastatic breast cancer. The primary objective was to determine whether the combination produced a response rate of at least 50%.

Patients And Methods: Fifty-four patients with metastatic breast cancer received doxorubicin (60 mg/m(2) by intravenous [IV] injection) followed 1 hour later by docetaxel (60 mg/m(2) by IV infusion over 1 hour) every 3 weeks for up to eight cycles.

The reduced folate carrier in L1210 murine leukemia cells is a 58-kDa protein.

The reduced folate carrier (RFC1) is a major transporter for both natural reduced folates and antifolate chemotherapeutics. Using polyclonal antibodies targeted to epitopes at the loop between the sixth and seventh predicted transmembrane domains or the distal C-terminus, we were able to demonstrate by Western blot analysis that the molecular size of RFC1 expressed in murine leukemia L1210 cells is 58 kDa as predicted by the open reading frame of its cDNA. 46- and 38-kDa proteins detected only in plasma membrane preparations were proteolytic degradation products that appeared during membrane preparation or treatment with the conventional SDS-PAGE loading buffer.

Role of the amino acid 45 residue in reduced folate carrier function and ion-dependent transport as characterized by site-directed mutagenesis.

In previous reports, an E45K mutation in reduced folate carrier (RFC1) resulted in marked substrate-specific changes in folate binding and the induction of an obligatory inorganic anion requirement for carrier function. In this study, site-directed mutagenesis was employed to further characterize the role of glutamate-45 in carrier function by replacement with glutamine, arginine, aspartate, leucine, or tryptophan followed by tranfection of the mutated cDNAs into the MTX(r)A line, which lacks a functional endogenous carrier. Alterations in transport function with amino acid substitutions at this residue were not charge related.

Phase II trial of doxorubicin and paclitaxel plus granulocyte colony-stimulating factor in metastatic breast cancer: an Eastern Cooperative Oncology Group Study.

Purpose: Several groups have reported that the combination of doxorubicin plus paclitaxel given as a 3-hour intravenous (IV) infusion for up to eight cycles produces a high response rate (> 80%) and complete response rate (> 20%) in metastatic breast cancer, but is also complicated by a 20% incidence of congestive heart failure (CHF). The purpose of this phase II trial was to evaluate the antineoplastic activity of the regimen in a multi-institutional setting and to reduce the incidence of cardiotoxicity by limiting treatment to a maximum of six cycles.

Patients And Methods: Fifty-two patients with advanced breast cancer received doxorubicin (60 mg/m(2) by IV injection) followed 15 minutes later by paclitaxel (200 mg/m(2) by IV infusion over 3 hours) every 3 weeks for four to six cycles.

Patterns of central nervous system recurrence in patients with systemic human immunodeficiency virus-associated non-hodgkin lymphoma.

Background: Central nervous system involvement is a common manifestation of non-Hodgkin lymphoma (NHL) in human immunodeficiency virus (HIV)-infected individuals. The purpose of this study was to review the frequency and pattern of neurologic manifestation of lymphoma in a cohort of HIV-infected individuals with systemic NHL.

Methods: Sixty-two patients with HIV-associated systemic NHL received infusional cyclophosphamide, doxorubicin, and etoposide.

Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier.

A phenylalanine substitution for serine in the reduced folate carrier at residue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cell line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folate source. The transport characteristics of the mutated carrier were studied by transfection into the MTXrA line, which lacks endogenous RFC1 function. The level of expression of carrier in the cell lines studied was determined by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF).

Inhibitory effects of prostaglandin A1 on membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters.

Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A1 (PGA1) on processes that govern the transport of folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the reduced folate carrier, RFC1, and ATP-dependent exporters. PGA1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a Ki of approximately 21 microM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity.

Effect of highly active antiretroviral therapy on the incidence of HIV-associated malignancies at an urban medical center.

The widespread use of highly active antiretroviral therapy (HAART) since 1996 has led to a substantial decline in morbidity and mortality in patients infected with HIV, although its effect on the incidence of HIV-associated malignancies is unknown. We retrospectively reviewed the annual number of outpatient visits to our HIV clinic, inpatient admissions for HIV disease, and first admissions for patients with cancer and HIV disease at our center between 1990 and 1997. Between 1990 and 1995, there was a progressive increase in the annual number of admissions for HIV disease and HIV-associated cancers that paralleled the increasing HIV clinic volume.

Doxorubicin/taxane combinations: cardiac toxicity and pharmacokinetics.

Doxorubicin and the taxanes, paclitaxel and docetaxel (Taxotere; Rhône-Poulenc Rorer, Collegeville, PA), are among the most active cytotoxic agents for the treatment of metastatic breast cancer. Given their activity, relative non-cross-resistance, partially non-overlapping toxicities, and differing mechanisms of action, there is a clear rationale for combining these agents for both advanced and early stage disease. Phase I and II trials have been reported for both doxorubicin/paclitaxel and doxorubicin/docetaxel.

Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis.

Chemical mutagenesis with N-methyl-N-nitrosourea was employed to study the pattern of mutations in the reduced folate carrier (RFC1) that results in transport-related methotrexate resistance and to identify amino acid residues that are critical to carrier structure and/or function. Thirty-four methotrexate transport-defective L1210 leukemia cell lines were isolated with folic acid as the sole folate source under antifolate selective pressure. The RFC1 mRNA levels were comparable with, or not substantially decreased, in most of these cell lines relative to wild-type L1210 cells.

Phase I trial of escalating doses of paclitaxel plus doxorubicin and dexrazoxane in patients with advanced breast cancer.

Purpose: To determine the maximum-tolerable dose (MTD) of paclitaxel given as a 3-hour intravenous (IV) infusion that could be used in conjunction with doxorubicin and dexrazoxane, and to determine the effect of dexrazoxane on the pharmacokinetics of paclitaxel and doxorubicin.

Patients And Methods: Twenty-five patients with advanced breast cancer received dexrazoxane (600 mg/m2 by IV infusion over 15 minutes), followed 15 minutes later by doxorubicin (60 mg/m2 IV), followed 15 minutes later by paclitaxel (150 or 175 mg/m2 by IV infusion over 3 hours) in cohorts of three to six patients using a standard phase I design without (group A) and with (group B) granulocyte colony-stimulating factor (G-CSF). Treatment continued until there was a substantial decrease in the left ventricular ejection fraction (LVEF), congestive heart failure, progressive disease, or physician discretion to discontinue.

A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function.

In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively.

Concentrating capacity of the human reduced folate carrier (hRFC1) in human ZR-75 breast cancer cell lines.

Human RFC1 (hRFC1) transfected in transport-deficient methotrexate MTXR(R)ZR-75-1 human breast carcinoma cells (MTX(R)ZR-75/RFC) were used to investigate the impact of hRFC1 overexpression on influx and concentrative transport of methotrexate (MTX). Eight-fold overexpression of hRFC1, as determined by northern analysis, resulted in a 4-fold increase in MTX influx accompanied by a 2.4-fold increase in the steady-state level of free drug as compared with wild-type ZR-75-1 cells when the extracellular MTX level was 0.