5-methyltetrahydrofolate and Carcinoma--Squamous-Cell

To analyze clinical and pharmacokinetic data of cisplatin (CP)/fluorouracil (FU)/l folinic acid (l FA) chemotherapy administered as first-line treatment to locally advanced head and neck cancer patients.. Thirty-nine patients (35 men and four women; median age, 60 years; six stage III and 33 stage IV) received CP on day 1 (100 mg/m2) followed by l FA (200 mg/m2/d x 5) plus FU (500 mg/m2/d x 5) administered by continuous venous infusion (three cycles planned). Mean plasma concentrations of FU, l FA, and 5-methyltetrahydrofolate (5MTHF) over the cycle were computed.. Clinical response was assessable for 33 patients. Response rates on the primary tumor site (n = 33) were 63.7% complete responses (CRs), 24.2% partial responses (PRs), and 12.1% treatment failures. Response rates on lymph nodes (n = 27) were 40.7% CRs, 37.1% PRs, and 22.2% treatment failures. The most frequent toxicity was mucositis (36.2% of cycles grade 3 to 4). Grade 3 to 4 nausea-vomiting, diarrhea, neutropenia, and thrombocytopenia occurred in 6.7%, 1.9%, 13.3%, and 1% of cycles, respectively. Pharmacokinetic analysis showed a wide interpatient variability for both FU (mean, 1.01 mumol/L; range, 0.16 to 2.09), l FA (mean, 1.89, mumol/L; range, 0.52 to 7.88) and 5MTHF plasma concentrations (mean, 3.85 mumol/L; range, 1.30 to 8.11). A significant correlation was demonstrated between FU concentration and hematologic toxicity grade, mucositis grade, and nausea-vomiting/diarrhea grade. Regarding tumor response, patients who failed to respond significantly exhibited lower FU and total folate concentrations than patients with a CR or PR.. This study highlights the efficacy of CP/FU/l FA in head and neck carcinoma and establishes the clinical importance of coupled FU/FA pharmacokinetics to predict pharmacodynamic variability.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Female; Fluorouracil; Humans; Hypopharyngeal Neoplasms; Laryngeal Neoplasms; Leucovorin; Male; Middle Aged; Nose Neoplasms; Oropharyngeal Neoplasms; Tetrahydrofolates

UM-SCC-38 cells, a squamous cell carcinoma cell line of the head and neck, express limited amounts of folate receptor alpha antigen which is not capable of binding either folic acid or 5-methyltetrahydrofolic acid. Three distinct mutations in the open reading frame of the folate receptor were identified. We now show that the three mutants are nonfunctional with respect to folic acid binding because the protein products do not bind folate. Additionally, a study of MA104 cells (a receptor-positive cell line) transfected with each mutant was done. Expression of one mutant, FR-67, results in a dominant negative phenotype because folate binding is significantly reduced although membrane antigen is significantly increased. Coexpression of FR-67 and the normal protein in MA104 cells also results in large, bright clusters of receptor protein inside the cell around the nucleus when visualized using indirect immunofluorescence. These clusters are not found in cells that express either normal or FR-67 protein alone. In conclusion, this study provides the first evidence of a mutant folate receptor protein capable of affecting normal receptor function in a dominant negative manner.

Topics: Animals; Carcinoma, Squamous Cell; Carrier Proteins; CHO Cells; Cricetinae; DNA, Complementary; Folate Receptors, GPI-Anchored; Folic Acid; Genes, Dominant; Glycosylphosphatidylinositols; Haplorhini; Humans; Phenotype; Point Mutation; Receptors, Cell Surface; Tetrahydrofolates; Transfection; Tritium

Some cells accumulate folate via a receptor-coupled process termed potocytosis. The folate receptor, a glycosyl phosphatidylinositol anchored M(r) 38,000-39,000 glycoprotein, is coded for by at least two genes (FR alpha and FR beta) at 11q13. The karyotype of UMSCC38, a human squamous cell carcinoma cell line, suggests that it may contain multiple copies of the folate receptor gene(s). Southern blot analysis confirms the presence of four to six copies. Using polymerase chain reaction methodology, Northern blot analysis, immunoblotting, and immunofluorescence, we find relatively limited expression of FR alpha and no FR beta in UMSCC38 cells when compared to nonamplified lines. Antigen is observed on the cell surface in a punctate pattern, and the protein is anchored via a glycosyl phosphatidylinositol anchor. Transport of 5-[methyl-3H]tetrahydrofolic acid is blocked by 5-methyltetrahydrofolic acid and probenecid, which suggests anion transport. Monensin, an inhibitor of potocytosis, and folic acid, a high-affinity ligand for the receptor, do not effectively block 5-methyltetrahydrofolic acid transport. Taken together, the results suggest that UMCSCC38 cells, although gene amplified, synthesize surprisingly small amounts of receptor and that receptor is nonfunctional. In order to establish further the nature of the receptor, 16 clones were obtained, and the complementary DNA was sequenced. Three mutations were found.

Topics: Base Sequence; Biological Transport; Carcinoma, Squamous Cell; Carrier Proteins; Chromosomes, Human, Pair 11; DNA, Complementary; Folate Receptors, GPI-Anchored; Folic Acid; Gene Amplification; Humans; Molecular Sequence Data; Mutation; Receptors, Cell Surface; Tetrahydrofolates; Tumor Cells, Cultured

To study the activity of continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin (PFL) as induction chemotherapy in patients with previously untreated, advanced squamous cell carcinoma of the head and neck.. Nonrandomized, prospective trial.. A comprehensive cancer center.. Thirty-five patients (4 patients [11%], stage III; 31 patients [89%], stage IV [MO]), all evaluable for response and toxicity.. Two to three cycles of PFL before definitive, local-regional therapy (surgery and radiation therapy or radiation therapy alone). Chemotherapy included continuous intravenous infusion of cisplatin (25 mg/m2 body surface area daily, days 1 through 5); 5-fluorouracil (800 mg/m2 body surface area daily, days 2 through 6); and leucovorin (500 mg/m2 body surface area daily, days 1 through 6) administered once every 28 days. Pathologic response was evaluated by surgical resection or biopsy. Serum-reduced folates were measured before and 18 hours after the initiation of chemotherapy.. A clinical response to PFL was achieved in 28 of 35 (80%) patients: 23 (66%) patients had a complete response (90% CI, 50% to 79%) and 5 (14%) patients, a partial response. A complete response was confirmed pathologically in 14 of 19 (74%) patients. The most common toxicity was mucositis (grade 2 to 3; 94% of patients). Dose reduction for toxicity was necessary in 11 (31%) patients. There were no treatment-related deaths. Serum levels of leucovorin and (6S)5-methyltetrahydrofolate were measured in 7 patients. After 18 hours, the mean leucovorin level (+/- SD) was 34.3 +/- 1.5 mumol/L, of which only 8.0 +/- 0.5% was the active 6S isomer. The mean serum (6S)5-methyltetrahydrofolate was 9.2 +/- 0.6 mumol/L.. Continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin is a new and highly active chemotherapy regimen that can achieve clinical and pathologically confirmed complete responses in a substantial proportion of patients with advanced, local-regional squamous cell carcinoma of the head and neck. Further studies are needed to confirm the activity of PFL and to determine its potential impact on local tumor control and disease-free and overall survival.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Drug Administration Schedule; Drug Evaluation; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Infusions, Intravenous; Leucovorin; Male; Middle Aged; Neoplasm Staging; Tetrahydrofolates

Human nasopharyngeal epidermoid carcinoma (KB) cells contain a membrane-associated particulate folate-binding protein which is important in the cellular accumulation of physiologic folates (Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, P. C., and Kolhouse, J. F. (1985) J. Biol. Chem. 260, 14911-14917) and in the binding of methotrexate (Kane, M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) J. Biol. Chem. 261, 44-49). A soluble folate-binding protein appears in media exposed to proliferating KB cells. We have purified to homogeneity both the membrane-associated and the soluble folate-binding proteins from the KB cell tissue culture system. The purified membrane-associated and soluble folate-binding proteins give single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr values of 50,000 and 40,000, respectively. The membrane-associated folate-binding protein contains 45,000 g of amino acids and the soluble folate-binding protein contains 24,000 g of amino acids per mole of folate bound. Each of the purified proteins has a single folate-binding site, and the carbohydrate content is approximately 25% for each species of protein. The affinity constants for 5-methyltetrahydrofolate of the membrane-associated and soluble folate-binding proteins are 0.3 and 2.5 X 10(9) liters/mol, respectively. The affinities of various polyglutamated forms of methotrexate are similar for each protein, increase as the chain length of the polyglutamate increases (from approximately 0.004 X 10(9) liters/mol for methotrexate to 0.3 X 10(9) liters/mol for methotrexate heptaglutamate), are equal to the affinity for 5-methyltetrahydrofolate, and exceed the reported increase in affinity of methotrexate polyglutamates for dihydrofolate reductase.

Topics: Amino Acids; Carbohydrates; Carcinoma, Squamous Cell; Carrier Proteins; Cell Membrane; Chromatography, Gel; Cytosol; Electrophoresis, Polyacrylamide Gel; Folate Receptors, GPI-Anchored; Humans; KB Cells; Methotrexate; Milk, Human; Molecular Weight; Nasopharyngeal Neoplasms; Polyglutamic Acid; Receptors, Cell Surface; Tetrahydrofolates

The characteristics of the uptake by human epidermoid carcinoma (KB) cells of 5-methyltetrahydrofolate at extracellular concentrations in the physiologic range and the possible role of a membrane-associated folate binder in folate uptake by KB cells have been investigated. Uptake of 5-methyltetrahydrofolate was specific, saturable, and time-, temperature-, and concentration-dependent. Trypsin treatment released 50% of the 5-methyltetrahydrofolate accumulated by KB cells at 4 degrees C, but only 12% at 37 degrees C, indicating that most of the accumulated ligand was intracellular at 37 degrees C, thus demonstrating transport. Accumulated 5-methyltetrahydrofolate was bound to a membrane-associated protein which required detergent for its solubilization, and a significant amount of which was oriented to the cell exterior as demonstrated by its release by trypsin treatment of intact KB cells. The membrane-associated folate binder was immunoprecipitated by antiserum to purified human placental folate receptor, and this antiserum inhibited 5-methyltetrahydrofolate uptake by intact KB cells in a concentration-dependent manner. These data support the hypothesis that the membrane-associated folate-binding protein of human cells participates in the transport of folates under physiologic conditions.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Molecular Weight; Receptors, Cell Surface; Temperature; Tetrahydrofolates; Time Factors; Trypsin

Seventeen patients with heavily pretreated head and neck squamous cell carcinoma were submitted to a combination of 5-fluorouracil, 500 mg/m2 on days 1-4, and cis-platin, 50 mg/m2 on day 5, repeated every 21 days. Before administration of 5-fluorouracil, N5,N10-methyltetrahydrofolate, 200 mg/m2 i.v., was given. Only 1 partial response and 4 stable disease were observed, and the median survival of the entire group was 5 months. Although all patients had been heavily pretreated and a considerable percentage (6/17, 35.2%) of these showed resistance to first-line therapy, this combination seems to be ineffective as second-line therapy in head and neck cancer.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Tetrahydrofolates