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Biomedical subjects

D Banerjee

Publications and source records attributed to D Banerjee.

At least 127 records · Page 7Linked to original sources
PubMed1995

Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.

Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.

Antigens, CD↗
PubMed1995

High-dose continuous venous infusion of interleukin-2: influence of dose and infusion rate on tumoricidal function and lymphocyte subsets.

Previous clinical studies have demonstrated a dose-response relationship between enhancement of certain immune parameters and interleukin-2 (IL-2) dose in trials with low dosages of the cytokine. This has not been demonstrated for high-dose (greater than 18 x 10(6) IU/m2 per day) IL-2. We completed phase II trials of sustained administration of indomethacin and ranitidine with IL-2 given as a continuous infusion over 5 days for three courses. Peripheral blood mononuclear cells, both fresh and cultured in vitro with IL-2 or IL-2 and indomethacin, were tested for tumoricidal function against K562 and Daudi targets; these results were then correlated with actual delivered dose and mean infusion rate per course. Similar correlations were calculated between delivered dose or infusion rate and absolute and proportional counts of lymphocyte subsets as determined by flow cytometry. No enhancement of in vitro tumoricidal function with either increasing delivered dose or increasing infusion rate was seen. No consistent pattern of correlation was found between the absolute counts of lymphocyte subsets after each course of IL-2 with delivered dose or infusion rate. The percent rise in absolute counts of selected T- and NK-cell subsets at the end of course 1 compared with baseline values correlated positively with infusion rate; however, a similar correlated between the infusion rate and an increase in lymphocyte tumoricidal function was lacking. Little evidence was found for improved tumoricidal function of mononuclear cells or consistent enhancement of lymphocyte subset counts in patients able to tolerate doses of IL-2 beyond 18 x 10(6) IU/m2 per day in a 5-day continuous infusion schedule.

Carcinoma, Renal Cell↗
PubMed1995

Biodistribution and virus inactivation efficacy of a silicon phthalocyanine in red blood cell concentrates as a function of delivery vehicle.

The silicon phthalocyanine, HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc 4), is a new photosensitizer that can inactivate lipid-enveloped viruses in red blood cell concentrates (RBCC) upon exposure to red light. Because Pc 4 is insoluble in water, it was delivered either as an emulsion in saline and cremophor EL (CRM) or as a solution in dimethyl sulfoxide (DMSO). In RBCC, Pc 4 added in either vehicle distributed between the plasma and red blood cells (RBC) in a ratio of 4:6, similar to the ratio of these components in RBCC 3:7 (i.e. a hematocrit of 70%). Light exposure did not affect this distribution and caused only marginal degradation of Pc 4 at a light dose that inactivates > 5 log10 vesicular stomatitis virus (VSV). Among human plasma proteins, Pc 4 bound mainly (about 70%) to lipoproteins and to a lesser extent to albumin and lower molecular weight proteins when delivered in DMSO. When delivered in CRM, distribution between lipoproteins and albumin became more even. Among the lipoproteins Pc 4 bound almost exclusively to very low-density lipoproteins (VLDL) when delivered in DMSO and to both VLDL and low-density lipoproteins when added in CRM. The rate of VSV inactivation was independent of the delivery vehicle but there was less RBC damage, as measured by hemolysis during storage, when Pc 4 was added in CRM. These results indicate that using CRM as emulsifier can enhance the specificity of Pc 4-induced photochemical decontamination of RBCC for transfusion.

Animals↗
PubMed1995

Flow cytometry related to hematopoietic malignancy.

Flow cytometry is used in the workup of hematological malignancies to establish the immunophenotype of a given malignancy and/or to determine DNA ploidy and S-phase fractions. Flow cytometric data is used as an adjunct to routine morphology and can have diagnostic, confirmatory and prognostic relevance. Samples that can be analyzed include blood, bone marrow, lymph nodes, fine needle aspirates and effusions. DNA analysis can be performed not only on fresh and frozen tissue but also on archival material.

DNA, Neoplasm↗
PubMed1995

Increased resistance to methotrexate in human hematopoietic cells after gene transfer of the Ser31 DHFR mutant.

Retroviral gene transfer into hematopoietic cells has many experimental as well as clinical applications. Although transduction efficiency of retroviral vectors is higher than with conventional methods, selection of successfully transduced cells may become mandatory for efficient in vivo transfer. We have been evaluating a retroviral construct that meets the criteria for a clinically acceptable selection system. The Ser31 dihydrofolate reductase (DHFR) mutant confers resistance to methotrexate (MTX) due to decreased binding of the drug. while its enzymatic activity remains adequate for normal folate metabolism. Transduction of this vector into murine hematopoietic cells has been recently described and increase in MTX resistance could be observed. We investigated transduction of CD34-antigen positive subpopulations of hematopoietic progenitor cells and CD34-positive/CD38-negative subpopulations enriched for stem cells. We focused on two sources of primary hematopoietic cells, umbilical cord blood (UCB) and peripheral blood (PB) harvested from patients after mobilization with chemotherapy and/or cytokines. Both contain a large number of lineage restricted and pluripotent progenitor cells and can be expanded extensively ex vivo. Potential clinical applications of gene therapy in such cell populations include correction of inborn enzymatic diseases and support of high-dose chemotherapy by transplanting ex vivo transduced progenitor cells rendered more resistant to cytotoxic drugs. The feasibility and efficiency of retroviral transduction into UCB and PB has been reported recently.

Antigens, CD34↗
PubMed1995

Acute monocytic leukemia: a myeloid leukemia subset that may be sensitive to methotrexate.

Impaired polyglutamylation of methotrexate (MTX) and thus poor retention is believed to be the basis of intrinsic resistance in blasts from patients with acute myeloid leukemia (AML) to MTX. We studied additional samples from patients with this disease, and confirmed that polyglutamylation of MTX was poor in ANLL blast cells. However, in one subset of ANLL, acute monocytic leukemia, (M5) leukemia blasts were found to be capable of accumulating and forming long-chain MTX polyglutamates. An acute monocytic leukemia cell line, THP-1 also was found to accumulate high levels of MTX polyglutamates and was relatively sensitive to MTX, strengthening the concept that M5 blasts may be sensitive to this drug. MTX may be an overlooked drug for the treatment of acute monocytic leukemia.

Adult↗
PubMed1995

Multiparameter flow cytometric DNA analysis of effusions: a prospective study of 36 cases compared with routine cytology and immunohistochemistry.

Single-parameter flow cytometry (SFCM) is limited in its ability to detect aneuploid and diploid malignant cells or accurately estimate S-phase fractions (SPF) in effusions because of the high degree of contamination by benign mesothelial cells and inflammatory cells. We examined 36 pleural and peritoneal fluids by conventional cytology and multiparameter FCM (MFCM) to analyze the DNA content of cells expressing epithelial markers cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, BRST-1, or BRST-3 (B72.3) and compared the results to those found with SCFM. The cases were also studied by immunohistochemistry using the same antibody panel. By routine cytology, 14 of the 36 cases were classified as carcinomas, 11 as reactive, 1 as mesothelioma, and 10 as suspicious. MFCM allowed reclassification of 5 of the 10 suspicious cases as carcinomas and the remaining 5 as reactive cases based on ploidy and marker expression. Whereas SFCM detected only 13 nondiploid carcinomas, MFCM detected 4 diploid and 15 nondiploid carcinomas. All reactive cases were diploid by SFCM or MFCM. The mesothelioma case showed were distinct peaks by MFCM, a diploid peak with SPF of 13.4% and a tetraploid peak with SPF of 36.1%. The SPF of the nondiploid carcinomas ranged from 5.9 to 50.4% and diploid carcinomas, from 3.5 to 14.5% when gated on epithelial cells. The reactive cases had SPF ranging from 0.4 to 4.4%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1995

Transfection of wild-type but not mutant p53 induces early monocytic differentiation in HL60 cells and increases their sensitivity to stress.

HL60 cells, which lack the p53 gene due to a deletion, were used as an in vitro model system to study the effect of wild-type p53 gene expression on hematopoietic differentiation. We transfected HL60 cells with wild-type p53 and two mutant p53 cDNAs encoding the Val to Ala mutation at codon 143 and the Arg to Trp mutation at codon 248. Flow cytometry, growth, and cytochemical analysis for alpha-napthyl butyrate esterase activity and nitroblue tetrazolium reduction indicated that wild-type p53 but not mutant p53 induced early monocytic differentiation in the transfected HL60 cells without terminal growth arrest. The wild-type p53 transfectants did not differentiate along the granulocytic pathway, even when induced with 1.25% DMSO for 6 days; rather, these cells resembled monocytic cells, confirming that wild-type p53 transfection caused these cells to become committed to differentiate along the monocytic pathway. HL60 cells transfected with wild-type p53 were more sensitive to stress, such as growth in serum-depleted medium and exposure to a chemotherapeutic agent, etoposide.

Antigens, Surface↗
PubMed1995

Cytogenetic study of primary amenorrhoea.

A study of chromosomal pattern was done in 60 cases of primary amenorrhoea of different age groups to determine the incidences of chromosomal abnormalities in them and to detect those cases of classical 45, X Turner's syndrome and Turner mosaics that do not bear the Turner stigmata. Buccal smears were examined for sex chromatin followed by karyotype using leucocyte culture method. Majority (63.3%) of cases were found chromosomally incompetent of which the major abnormality was 45, X/46, XX mosaicism (33.3%) followed by 45, X Turner's syndrome (26.6%). But only 43.7% of these Turner's syndrome had classical Turner stigmata. Two cases of complete testicular feminisation syndrome with male genotype (46,XY) and inguinal testis were also detected.

Adult↗
PubMed1995

Molecular mechanisms of resistance to antifolates, a review.

Methotrexate (MTX) is a clinically important antifolate that has been used in combination with other chemotherapeutic agents in the treatment of malignancies including acute lymphocytic leukemia, osteosarcoma, carcinomas of the breast, head and neck, choriocarcinoma and non-Hodgkin's lymphoma. The primary target of MTX is the enzyme dihydrofolate reductase (DHFR) which catalyzes the reduction of folate and 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Understanding of MTX action has revealed how cells acquire resistance to this drug. The four known mechanisms of MTX resistance are a decrease in the uptake of the drug, a decrease in the retention of the drug due to defective polyglutamylation or an increase in polyglutamate breakdown, an increase in the enzyme activity and a decrease in the binding of MTX to DHFR. The molecular basis for some of these mechanisms has been elucidated in MTX resistant cell lines; in particular the occurrence of gene amplification resulting in increased DHFR and point mutations resulting in altered DHFR with reduced affinity for MTX. Cloning of the human folylpolyglutamate synthase gene and the reduced folate transport gene have been reported recently and should facilitate the identification of the molecular basis of these resistant phenotypes. DHFR protein has been shown to regulate its synthesis by exerting an inhibitory influence on its own translation. Addition of MTX relieves this inhibition thus providing a possible molecular explanation for the rapid rise in DHFR activity noted in some cells after MTX administration. Alterations in genes involved in regulating the cell cycle such as cyclin D1 and the retinoblastoma (Rb) gene have also been shown to influence cellular response to MTX. Overexpression of cyclin D1 in HT1080, a human fibrosarcoma cell line, results in decreased MTX sensitivity. The molecular basis of this observation is under investigation. Abnormalities in the Rb gene may also have profound effects on MTX sensitivity. Rb interacts with the family of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR. When Rb is deleted or rendered nonfunctional levels of "free" or unbound E2F are high resulting in enhanced transcription of genes such as DHFR. This results in increased DHFR protein and may lead to MTX resistance. As the knowledge regarding mechanisms of resistance increases newer approaches to circumvent such resistance or to target resistant cells can be undertaken.

Antineoplastic Agents↗
PubMed1994

Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction.

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock-infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

3T3 Cells↗
PubMed1994

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Animals↗
PubMed1994

Gene therapy utilizing drug resistance genes: a review.

The generation of drug resistant bone marrow may facilitate the development of aggressive chemotherapeutic regimens that might otherwise be lethal due to marrow toxicity. With the availability of technology that permits in vitro manipulation of human marrow and peripheral blood stem cells, it is now possible to introduce genes that confer drug resistance to these hematopoietic progenitors. Animal models and in vitro work with human progenitors using drug resistance genes are reviewed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Nasopharyngeal carcinoma in the young.

This study was carried out to determine whether locoregional control of nasopharyngeal carcinoma in the young by irradiation has improved since 1975. Fifty-seven consecutive, previously untreated, patients, less than 30 years old, were diagnosed and treated at University of Toronto Hospitals between 1958 and 1990; 21 patients were treated before and 36 after 1975. Staging was as follows: M0, n = 54; M1, n = 3; T1 + T2, n = 26; T3 + T4, n = 31; N0, n = 10; N1 + N2a, n = 10; N2b + N2c, n = 24; N3, n = 13. All patients were irradiated. The primary tumour dose was 3500-7000 cGy (median 5450). Adjuvant chemotherapy was introduced in 1977. Subsequently, 10/26 (38%) M0 patients received this treatment. For all 57 patients, 10-year survival was 56%. For 52 M0 patients with complete follow-up data, 10-year survival was 63%; 10-year relapse free survival (RFS) was 61%. Age, sex, race, and histology were not significant variables. Ten-year RFS results were: T1 + T2, M0 (n = 24) 70%, T3 + T4, M0 (n = 27) 52%, (P = 0.25); N0-N2c (n = 41) 64%, N3 (n = 11) 54% (P = 0.31). Relapse occurred in 20/52 (38%) patients. Survival from the date of first relapse was 10% at 10 years. No patient with systemic relapse survived. Only 1/32 (3%) patients treated after 1975 developed an isolated locoregional relapse, giving a 10-year isolated locoregional relapse free rate (LR RFS) of 96%, compared with 5/20 (25%) prior to 1975, which gave a 10-year LR RFS of 75% (P = 0.05). Two of the five patients who relapsed before 1975 were salvaged by re-irradiation. Systemic relapse, either alone or combined with locoregional relapse, accounted for 6/11 (55%) relapses before 1975 and 8/9 (89%) after 1975. Ten-year systemic RFS was 90% for patients who received adjuvant chemotherapy (n = 10) and 72% for patients treated since 1977 by irradiation alone (n = 16) (P = 0.41). Isolated local relapse was exceptional in patients treated after 1975 (1/32).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEOr gene and a mutated dihydrofolate reductase cDNA (Leu 22-->Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Primary, secondary, and even tertiary recipients transplanted with bone marrow cells infected with the recombinant virus showed protection from lethal methotrexate toxicity. The viral construct containing a SV-40 promoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deaminase promoter, although both afforded protection. Evidence for integration into blood cells of both the NEOr gene and the mutated dihydrofolate reductase gene was obtained by polymerase chain reaction; sequencing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoietic progenitor cells in the mouse can be successfully transduced with a drug resistance gene.

Animals↗