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Biomedical subjects

R Gorlick

Publications and source records attributed to R Gorlick.

At least 19 recordsLinked to original sources
PubMed1999

Mechanisms of methotrexate resistance in acute leukemia. Decreased transport and polyglutamylation.

Drug resistance limits the effectiveness of methotrexate (MTX) for the treatment of acute leukemia. An increased understanding of the pathways involved in folate metabolism has allowed investigations of the mechanisms of resistance observed in leukemic blasts obtained from patients. Acute lymphocytic leukemia (ALL) was studied for mechanisms of acquired MTX resistance. MTX transport in 27 patients with untreated ALL and 31 patients with relapsed ALL was measured using a previously described competitive displacement assay. Only 13% of the untreated patients were considered to have impaired MTX transport whereas over 70% of the relapsed patients had evidence of impaired MTX transport. Northern analyses and quantitative RT-PCR for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of 9 samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. Acute myelocytic leukemia (AML) was studied to determine the basis of the decreased MTX polyglutamylation. Enzyme kinetics of the enzyme folylpolyglutamate synthetase (FPGS) were studied, demonstrating FPGS in the myeloid cell lines and patient samples had a higher K(m) for MTX as a substrate than lymphoid cells. Measuring gamma-glutamyl hydrolase enzyme activity allowed a more accurate prediction of steady state levels of MTX polyglutamates. A knowledge of the mechanisms of MTX resistance that occur in leukemic blasts obtained from patients may allow the development of therapeutic strategies to circumvent resistance.

Antimetabolites, Antineoplastic↗
PubMed1999

Expression of bone morphogenetic proteins and receptors in sarcomas.

Bone morphogenetic proteins, which are capable of inducing mesenchymal tissue to form bone in mammals, have been implicated as important in normal skeletal development. The expression of bone morphogenetic proteins and their receptors were studied in 36 osteosarcoma specimens, six Ewing's sarcomas, 20 synovial sarcomas, and 20 chondrosarcomas by reverse transcriptase-polymerase chain reaction, and the findings were correlated with clinical data. Bone morphogenetic protein-2, and -4 messages were detected in most sarcoma samples. Bone morphogenetic protein-6 expression was detected in 22 of 32 osteosarcomas and seven of eight chondrosarcomas. Bone morphogenetic protein-7 and receptor IB were not detected in sarcoma samples but were detected in three osteosarcoma cell lines and one malignant fibrous histiocytoma cell line. Expression of bone morphogenetic protein receptor II was found in 25 of 36 osteosarcomas, eight of 20 chondrosarcomas, four of six Ewing's sarcomas, and 15 of 20 synovial sarcoma samples. Expression of bone morphogenetic protein type II receptor was found to correlate with metastasis in osteosarcomas, which suggests that the bone morphogenetic protein pathway may participate in tumor aggressiveness or progression. The expression of bone morphogenetic protein receptor II in metastatic synovial sarcoma and dedifferentiated chondrosarcoma lesions also supports this hypothesis. The current study showed that the ligands for bone morphogenetic protein receptors, bone morphogenetic proteins-2, -4, and -6 also are expressed in osteosarcoma and other sarcoma tissues, indicating a potential for autocrine or paracrine growth stimulation in these tumors.

Adult↗
PubMed1999

Expression of HER2/erbB-2 correlates with survival in osteosarcoma.

PURPOSE: In osteosarcoma, prognostic factors at diagnosis other than clinical stage have not been clearly identified. The aim of this study was to determine whether human epidermal growth factor receptor 2 (HER2)/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or event-free survival. PATIENTS AND METHODS: We performed a retrospective immunohistochemical study on material obtained from patients treated on the Memorial Sloan-Kettering Cancer Center T12 protocol between 1986 and 1993. Paraffin-embedded tissue was identified from 53 patients (73% of patients enrolled onto protocol) and stained for HER2/erbB-2, p53, and p-glycoprotein expression using standard monoclonal antibodies and methods. RESULTS: At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Higher frequencies of expression were observed in samples from patients with metastatic disease at presentation and at the time of relapse. Expression of HER2/erbB-2 correlated with a significantly worse histologic response (P =.03). In patients presenting with nonmetastatic disease, expression of HER2/erbB-2 at the time of initial biopsy was associated with a significantly decreased event-free survival (47% v 79% at 5 years, P =.05). p53 and p-glycoprotein expression did not correlate with histologic response or patient event-free survival. CONCLUSION: The correlation of HER2/erbB-2 expression with histologic response to preoperative chemotherapy and event-free survival in this study suggests that HER2/erbB-2 should be evaluated prospectively as a prognostic indicator. The correlation also suggests that clinical trials of antibodies that target this receptor, such as recombinant humanized anti-HER2 monoclonal antibody (Herceptin; Genentech, San Francisco, CA), should be considered for the treatment of osteosarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Mechanisms of methotrexate resistance in osteosarcoma.

High-dose methotrexate is a major component of current protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resistance include decreased transport through the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantitative reverse transcription-PCR. The RFC and DHFR genes were studied for deletions and amplification by Southern blot. Thirteen of 20 (65%) osteosarcoma samples were found to have decreased RFC expression at the time of initial biopsy. At definitive surgery and relapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (P = 0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at initial biopsy. The frequency of increased DHFR expression was significantly higher in metastatic or recurrent tumors (62%, P = 0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy material suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions.

Adolescent↗
PubMed1999

Drug resistance in colon cancer.

A significant obstacle for the successful management of patients with colorectal cancer is intrinsic drug resistance or, in patients who respond to chemotherapy, acquired drug resistance. Drug resistance can occur through a variety of mechanisms, including alterations in drug influx, drug efflux, intracellular metabolic activation, and intracellular catabolism, or through alterations in the drug's target. In addition, alterations in genes involved in the regulation of the cell cycle or in DNA damage repair may result in a cell becoming resistant to chemotherapy. In this chapter, the mechanisms of action and the mechanisms of resistance to the fluoropyrimidines and raltitrexed (Tomudex; Zeneca Pharmaceuticals, Wilmington, DE) are reviewed, focusing on newer studies using gastric and colorectal tumor samples obtained from patients. Clinical trials using this new information are anticipated.

Animals↗
PubMed1998

Higher levels of thymidylate synthase gene expression are observed in pulmonary as compared with hepatic metastases of colorectal adenocarcinoma.

PURPOSE: It has been observed previously that the pulmonary metastases of colorectal adenocarcinoma are less responsive to therapy with fluorouracil (FUra) as compared with other sites of metastasis (liver, local). To investigate the basis of this chemoresistance, the levels of thymidylate synthase (TS) mRNA and protein were measured, as TS expression has been shown to be predictive of response to therapy in colorectal cancer. MATERIALS AND METHODS: Tumors were obtained from 19 patients with metastatic colorectal cancer (12 hepatic and seven pulmonary). TS expression was measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and TS protein levels were measured by Western blotting. The presence of TS amplification was assessed by Southern blotting. Levels of p53 protein were determined using immunohistochemistry. RESULTS: TS mRNA expression was shown to be significantly higher in the pulmonary metastases (mean TS/beta-actin ratio, 19.7; n = 7) as compared with the hepatic metastases (mean TS/beta-actin ratio, 4.7; n = 11) of colorectal cancer. Lower TS expression was observed in patients with hepatic metastases who had received prior FUra versus patients who had not been treated. High levels of TS expression in some samples was associated with low-level (two to three gene copies) increases in TS gene copy numbers and this was observed more frequently in the pulmonary metastatic samples. The increased gene copy numbers occurred both in samples with wild-type p53 and those with mutant p53 tumor-suppressor gene as determined by immunohistochemistry. CONCLUSION: High levels of TS enzyme may be the basis of the lack of response of pulmonary metastases to FUra treatment.

Adenocarcinoma↗
PubMed1998

Intensification of preoperative chemotherapy for osteogenic sarcoma: results of the Memorial Sloan-Kettering (T12) protocol.

PURPOSE: It has been observed previously in osteosarcoma (OS) that the degree of necrosis of the resected primary tumor following a period of preoperative chemotherapy is predictive of subsequent event-free survival (EFS). The aim of this study was to determine if more intensive preoperative chemotherapy would increase the proportion of patients with a good histologic response and improve EFS. PATIENTS AND METHODS: Seventy-three patients with OS were treated at Memorial-Sloan Kettering Cancer Center (MSKCC) on the T12 protocol between 1986 and 1993. Patients were randomized between therapy based on the T10 protocol and therapy with more intensive preoperative chemotherapy. The more intensive preoperative regimen consisted of two courses of cisplatin (CDDP) and doxorubicin (DOX) in addition to the usual preoperative regimen of high-dose methotrexate (HD MTX) and bleomycin, cyclophosphamide, and dactinomycin (BCD). RESULTS: The regimen with more intensive preoperative chemotherapy achieved a modest increase in the proportion of patients with a good histologic response (44% with a grade III or IV histologic response v 37% in the control arm, 33% with grade IV histologic response v 13% in the control arm). EFS continued to correlate with histologic response. The actuarial 5-year EFS in patients with localized disease was 78% for the regimen with more intensive preoperative chemotherapy and 73% for the control arm. CONCLUSION: Despite modest increases in the proportion of patients with good histologic response with intensified preoperative chemotherapy, no improvement in EFS was observed.

Adolescent↗
PubMed1998

Pretreatment of colon carcinoma cells with Tomudex enhances 5-fluorouracil cytotoxicity.

The cytotoxic effect of sequence and dose of Tomudex (TX) and 5-fluorouracil (FUra) on an HCT-8 colon carcinoma cell line using a clonogenic assay was evaluated. Synergistic cell kill was obtained with 24 h of exposure to TX followed by 4 h of exposure to FUra. Marginal synergy was obtained with the same sequence but with a 5-day exposure to FUra. The reverse sequence, FUra (either 4 h or 5 days), followed by TX (24 h), resulted in less-than-additive cell kill. The synergistic effect was not due to augmented inhibition of thymidylate synthase, as determined by the measurement of thymidylate synthase activity by tritium release from [5-3H]2'-deoxyuridine. Surprisingly, an increase in intracellular levels of phosphoribosylpyrophosphate was observed after 24 h of exposure to TX, suggesting the possibility of an indirect effect of TX and/or its polyglutamates on purine biosynthesis. Moreover, we observed an increased formation of FUra nucleotides in the cells preexposed to TX, likely due to the increased intracellular levels of phosphoribosylpyrophosphate, that as a consequence led to an enhanced incorporation of FUra into RNA and increased cell killing.

Adenocarcinoma↗
PubMed1997

Disparate affinities of antifolates for folylpolyglutamate synthetase from human leukemia cells.

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.

Acute Disease↗
PubMed1997

Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression.

Methotrexate (MTX) transport was examined in 27 patients with untreated acute lymphocytic leukemia (ALL) and 31 patients with relapsed ALL using a previously described fluorescent MTX analog (PT430) displacement assay (Blood 80:1158, 1992). Only 13% of untreated patients were considered to have impaired MTX transport, whereas more than 70% of relapsed patients had evidence of impaired MTX transport. To further characterize the basis for this defect, Northern analyses for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of nine samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. None of 15 samples with normal MTX transport had decreased RFC expression. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed to quantitate RFC mRNA expression more accurately. Decreased RFC expression was demonstrated in six of the nine samples with impaired MTX transport, confirming the results obtained by Northern blot. These data indicate decreased RFC expression associated with impaired MTX transport is observed in relapsed ALL following treatment with MTX-containing therapy.

Adolescent↗
PubMed1997

Osteosarcoma.

Over the last several decades, significant advances have been made in our ability to understand and treat osteosarcoma. In this article we describe the diagnosis, evaluation, and treatment of patients with this disease. The surgical issues are discussed. We review the major clinical trials that have led to our current level of understanding. The current studies for the treatment of osteosarcoma are described.

Age Distribution↗
PubMed1997

Increased methotrexate polyglutamylation in acute megakaryocytic leukemia (M7) compared to other subtypes of acute myelocytic leukemia.

Acute myelocytic leukemia (AML) is a malignancy that is intrinsically resistant to methotrexate (MTX). AML blasts, when incubated with radiolabeled MTX, form lower amounts of long chain polyglutamates compared to acute lymphocytic leukemia (ALL) blasts, thus providing an explanation for their lack of responsiveness to MTX. Leukemic blasts obtained from two children with acute megakaryocytic leukemia (M7 subtype) when incubated with radiolabeled MTX showed increased accumulation of total as well as long chain MTX polyglutamates, comparable to levels previously demonstrated in another subtype of AML, acute monocytic leukemia (M5), as well as in blasts from patients with pre-B ALL. We suggest that M7-AML patients with blasts showing increased MTX polyglutamylation might benefit from treatment with MTX.

Adolescent↗
PubMed1997

gamma-Glutamyl hydrolase and folylpolyglutamate synthetase activities predict polyglutamylation of methotrexate in acute leukemias.

Decreased methotrexate (MTX) long-chain polyglutamate formation is associated with MTX resistance whereas high levels of MTX polyglutamate accumulation are found in the blasts of leukemia patients who respond to therapy and have improved outcome. The steady-state level of long-chain MTX polyglutamates depends on the balance of activities of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamates to MTX in a gamma-carboxyl linkage, and gamma-glutamyl hydrolase (GGH) or conjugase, which sequentially removes the terminal glutamate residue of MTX polyglutamates. FPGS and GGH activities as well as the formation of total and long-chain MTX polyglutamates were measured after incubation with [3H]MTX in 15 blast samples from patients with acute leukemias (myeloid and lymphoid). The ratio between GGH and FPGS activities was better at predicting the amount of polyglutamate accumulated in the 24-h [3H]MTX assay compared to the determination of either activity alone. The linear regression curve relating the relative levels of long-chain polyglutamates/total polyglutamates with the ratio of GGH/FPGS showed an r value of 0.81 (P < 0.001). These data suggest that the evaluation of both these enzymes at diagnosis may be used as a predictor of MTX polyglutamylation and therefore for response to MTX therapy and outcome.

Acute Disease↗
PubMed1996

Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line.

BACKGROUND: Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas. PURPOSE: This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line. METHODS: Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay. RESULTS: Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine. CONCLUSION: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents. IMPLICATIONS: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.

Antimetabolites, Antineoplastic↗
PubMed1996

Resistance mechanisms to methotrexate in tumors.

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Antimetabolites, Antineoplastic↗
PubMed1996

Resistance Mechanisms to Methotrexate in Tumors.

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Journal Article↗