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Biomedical subjects

D Banerjee

Publications and source records attributed to D Banerjee.

At least 19 recordsLinked to original sources
PubMed1998

Isolation and characterization of thymitaq (AG337) and 5-fluoro-2-deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells.

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex(R), BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drug-resistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC50 values 40- and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.

Cloning, Molecular↗
PubMed1998

Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA.

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.

Antigens, CD34↗
PubMed1998

Nasoethmoid schwannoma with intracranial extension. Case report and review of literature.

Schwannomas are very rare in the nose and paranasal sinuses; their presence both intra-and extracranially is still rarer. Here we present a case of nasoethmoid schwannoma with intracranial extension into anterior cranial fossa. Clinical, radiological, pathological and operative findings are discussed and the literature is reviewed. We recommend bifrontal craniotomy for removal of intracranial and nasoethmoid extensions of this tumor.

Adult↗
PubMed1998

Recurrence of kala-azar after PKDL: role of co-factors.

Recurrence of kala-azar after post kala-azar dermal leishmaniasis (PKDL) has remained uncommon. We report here two patients with recurrence of kala-azar (KA) after development of PKDL. In one case the second attack of KA was preceded by repeated attacks of malaria and tuberculosis, and in the other the recurrence of KA followed an attack of measles. While measles has earlier been suggested as co-factor in inducing transformation from sub-clinical to clinical kala-azar, malaria was demonstrated to enhance the virulence and invasiveness of Leishmania in an experimental model as well as under natural condition. We propose that in our cases, measles and repeated attacks of malaria or tuberculosis led to immunosuppression and recurrence of visceral leishmaniasis (VL).

Adolescent↗
PubMed1998

Magnetic resonance image detection of coincidental sphenoid sinus aspergillosis and pituitary microadenoma: a potential surgical disaster.

While localizing the microadenoma in a patient with Cushing's disease on magnetic resonance imaging (MRI), fungal granuloma of the sphenoid sinus was coincidentally detected. This helped in the proper management of sphenoidal disease before undergoing trans-sphenoidal removal of the microadenoma, thus averting the possible catastrophe of direct intracranial spread of aspergillus infection.

Adenoma↗
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

The prognostic role of p53, metallothionein, P-glycoprotein, and MIB-1 in muscle-invasive urothelial transitional cell carcinoma.

Tissue from primary tumors was analyzed for 118 patients with urothelial cancer who subsequently received cisplatin-based chemotherapy. Immunohistochemical staining was performed for nuclear p53 reactivity; for two proposed mediators of drug resistance, metallothionein (MT) and P-glycoprotein; and for the cell proliferation marker MIB-1. For each marker, immunoreactivity was expressed as a percentage of positively staining cells, and overall intensity of staining was graded on a scale from 0 to 3. The product of these two measurements was calculated to generate a percentage-intensity index. Clinical data were obtained independently via retrospective chart review. Chemotherapy regimens containing cisplatin (cisplatin, methotrexate, and vinblastine or methotrexate, vinblastine, doxorubicin, and cisplatin) were administered for metastatic disease (n = 64), for locally advanced disease (n = 45), or as an adjuvant treatment (n = 9). The overall response rate was 56% among 99 evaluable patients, and median survival was 12.7 months. By univariate analysis, Eastern Cooperative Oncology Group performance status (P = 0.0025), tumor grade (P = 0.03), percentage of MT staining (P = 0.01), and percentage-intensity index of MT staining (P = 0.04) were significant predictors of response to chemotherapy. The first three of these were significant in a multivariate model (P = 0.05, 0.04, and 0.04, respectively). By subgroup analysis, the percentage of MT staining predicted for response in metastatic disease (P = 0.03), but not in locally advanced disease (P = 0.28). Only performance status was significantly related to overall survival (P = 0.0001, log-rank test) in the whole cohort. Overexpression of MT in the 64 patients with metastatic disease was associated with a shorter survival (P = 0.04). Expression of p53, P-glycoprotein, and MIB-1 did not predict for survival. In conclusion, overexpression of MT is associated with a poorer outcome from chemotherapy, possibly due to cisplatin resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival.

Recent studies suggest that there may be a strong correlation between the p53 status of a tumor and a patient's response to chemotherapy. Therefore, we determined p53 status in 36 patients with disseminated colorectal cancer by cDNA sequencing and immunohistochemical staining, as well as by the gene expression level of thymidylate synthase (TS), the target enzyme of 5-fluorouracil (5-FU), by reverse transcription-PCR. Ten patients (28%) experienced a clinical response to 5-FU chemotherapy. Overall, TS expression and response to chemotherapy were associated: 9 of 18 (50%) patients with TS < or = 3.0 x 10(-3) responded, compared to 1 of 18 (6%) patients with TS > 3.0 x 10(-3) (P = 0.003). p53 mutations were found in 21 of 36 patients (58%) using cDNA cycle sequencing, and p53 protein overexpression was found in 20 of 32 patients (62%) using immunohistochemistry staining. Overall p53 status and response to chemotherapy were associated: 5 of 10 (50%) patients with wild-type p53 or negative p53 staining experienced a response, but only 5 of 26 (19%) patients with mutant p53 or p53 overexpression responded. TS expression, but not expression of p53, was significantly associated with overall survival (P = 0.002). Patients with wild-type p53 had significantly lower TS levels compared to patients with mutated p53 (P = 0.044). In this study, we also present data linking specific p53 point mutations to TS expression levels and resistance to 5-FU. Although the number of patients is relatively small, these results identify p53 status and TS gene expression as associated with response in disseminated colorectal cancer; independent studies are needed to confirm these findings and to provide information leading to a better understanding of the role of 5-FU-based chemotherapy in the treatment of colorectal cancer.

Adult↗
PubMed1998

Restoration of wild-type p53 activity in p53-null HL-60 cells confers multidrug sensitivity.

HL-60 cells that stably express transfected wild-type (wt) p53 were used to determine whether restoration of wt p53 increased the chemosensitivity of cells that normally lack p53 activity. The wt p53 HL-60 transfectants (SN3 cells) were more sensitive than the parental (S) cells to a number of common anticancer drugs representing various mechanisms of action, whereas HL-60 cells transfected with p53 genes mutated at codons 248 and 143 were not sensitized. The sensitization ratio due to the transfected wt p53 varied from about 2-fold for cisplatin to over 50-fold for thymidine. Cells treated with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were used to study changes in various p53-associated gene expressions. A higher percentage of apoptotic cells among the SN3 cells was observed than among the S cells at each concentration of FdUrd. The S cells had undetectable levels of bax and high levels of bcl-2, whereas the SN3 cells had undetectable levels of bcl-2 levels and appreciable basal levels of bax. After FdUrd treatment of SN3 cells, both p53 and bax levels increased, but the induction of bax was faster than that of p53 and paralleled the appearance of apoptotic DNA laddering. FdUrd treatment induced p21 expression and increased the G1 fraction of the SN3 cells but did not induce p21 or change the phase distribution in the S cells. FdUrd treatment also induced the expression and phosphorylation of cyclin D1 in the SN3 cells but not in the S cells. These results show that transfected wt p53 confers multidrug sensitivity to HL-60 cells by re-adjustment of the expressions of apoptosis genes and displays other properties characteristic of endogenously originated wt p53.

Antineoplastic Agents↗
PubMed1997

Dihydrofolate reductase protein inhibits its own translation by binding to dihydrofolate reductase mRNA sequences within the coding region.

Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.

Base Sequence↗
PubMed1997

Rapid movement of newly synthesized chicken apolipoprotein AI to trans-Golgi network and its secretion in Madin-Darby canine kidney cells.

MDCK cells were utilized to study the biosynthesis and secretion of chicken apolipoprotein AI (apoAI). A full-length apoAI cDNA in an eukaryotic expression vector was transfected into a MDCK-TGG cell line, expressing a trans-Golgi network (TGN) marker (TGG), and stable clones expressing apoAI were selected. Pulse-chase and cell fractionation studies showed that, compared to gp 80 (an endogenous secretory protein), apoAI was rapidly transported from RER to Golgi complex within 5 min and released from the Golgi complex to the cell exterior by 30 min. Immunofluorescence and three-dimensional laser scanning confocal microscopy revealed that at steady state apoAI was predominantly localized in the TGN. Transferrin uptake experiments showed that apoAI, localized in the TGN, was derived primarily from biosynthetic and not from endocytic routes. ApoAI was secreted in a nonpolarized manner. We suggest that apoAI stays transiently in the TGN prior to its secretion and that the major events of apoAI biosynthesis in vivo and in MDCK cells are conserved. Possible mechanisms of rapid ER to Golgi transport and TGN localization of apoAI are discussed.

Animals↗
PubMed1997

Post-transplant methotrexate administration leads to improved curability of mice bearing a mammary tumor transplanted with marrow transduced with a mutant human dihydrofolate reductase cDNA.

To test the concept that protection of bone marrow progenitor cells via introduction of a drug resistance gene would allow larger and curative doses of chemotherapy to be administered, we used mice bearing a transplanted breast cancer as a model system. Mice bearing the E0771 tumor were treated with lethal doses of cyclophosphamide (CPA) and rescued from toxicity by administration of bone marrow transduced with a mutant dihydrofolate reductase (DHFR) cDNA (Ser-31) in a retroviral construct. Animals receiving marrow not transduced with mutant DHFR cDNA died from methotrexate (MTX) toxicity, whereas mice transduced with mutant DHFR cDNA containing marrow were able to tolerate MTX treatment post-transplant; 44% of the mice had no demonstrable tumor when sacrificed on day 52. Another control group of mice treated with CPA and transplanted but not treated with MTX post-transplant succumbed to tumor regrowth. These data provide a strong rationale for the use of drug resistance genes to protect marrow from drug toxicity because the increase in dose tolerated may result in an improved cure rate of chemosensitive tumors.

Animals↗
PubMed1997

Modulation of cytotoxicity of chemotherapeutic drugs by activated H-ras.

Cells from a single MCF-7 clone were transfected with an isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible construct containing activated human H-ras with a Gly12 --> Val12 mutation. Expression of H-ras was induced by the presence of IPTG with low background. MCF-7-ras clones were examined for sensitivity to a wide variety of drugs under both induced and non-induced conditions. When expression of the activated ras was induced, these clones showed markedly increased resistance to cisplatin and mitomycin C, moderately increased resistance to methotrexate and trimetrexate, and no increased resistance to other drugs including taxol, doxorubicin, and etoposide. A DNA fragmentation assay revealed that DNA in MCF-7-ras cells treated with cisplatin under induced conditions was intact, whereas extensive degradation of DNA occurred in similarly treated cells under non-induced conditions. This result, along with the fact that MCF-7-ras cells, upon induction of the activated H-ras, showed increased resistance to drugs that bind DNA, indicates that the activated H-ras may play a role in the DNA repair process.

Antineoplastic Agents↗
PubMed1997

Interobserver variation in prostate cancer Gleason scoring: are there implications for the design of clinical trials and treatment strategies?

A series of prostate cancer histological slides from 71 patients were used to measure the interobserver variation among three pathologists awarding a Gleason score. The study was prompted on account of the use of histological grade to stratify patients prior to randomization within two clinical trials currently recruiting at our centre, and a proposed study that would allocate treatment depending upon the score awarded. The pathologists were expected to award a score based upon their day to day experience, there being no consensus meeting before-hand to agree on the grey areas of the Gleason grading system. We used the kappa statistic to assess the level of agreement. This was calculated both for comparison of the raw scores awarded by the three observers, as well as the grouped scores corresponding to those groupings used for the purposes of stratification in the two trials. The extent of the interobserver variation (weighted kappa) for the raw scores (Gleason scores 2-10) was 0.16 to 0.29 and for the grouped scores (Gleason scores < or = 7 or > or = 8), kappa was 0.15 to 0.29. For the raw scores, the total agreement rate was 9.9% and the total disagreement 26.8%; for the grouped scores the total agreement rate was 43.7%. It is concluded that, despite this level of agreement there is no concern regarding stratification using the Gleason score, because of the subsequent randomization. However, using a reported Gleason score to determine treatment might be inappropriate. These data indicate the value of a central review process for pathology grading in clinical trials, especially where the treatment is directly affected by this information.

Adenocarcinoma↗
PubMed1997

Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene.

We have previously shown that transfer of a mutated dihydrofolate reductase (DHFR) confers resistance to methotrexate (MTX) to infected cells. We report herein the construction of a retrovirus vector, DC/SV6S31tk, which carries the herpes simplex virus thymidine kinase gene (HSVtk) as well as the mutated Serine 31 DHFR (S31) cDNA. 3T3 cells infected with DC/SV6S31tk are more resistant to MTX than cells infected with DC/SV6S31, which carries the S31 and Neo gene. In DC/SV6S31tk-infected cells, a fraction of cells (20-40%) were more resistant to MTX compared with DC/SV6S31-infected cells, and these cells survived a 5-day exposure to 200 microM of MTX. The mechanism of this augmented resistance is attributed to the salvage of thymidine by HSVtk, as the augmentation is reversed when dialyzed serum is used for cytotoxicity assays. The cells that survive high-dose MTX selection have high levels of expression of S31 DHFR and HSVtk, although copy numbers of the proviral sequences do not increase significantly. Transduction of cells with the DC/SV6S31tk vector also sensitizes cells to ganciclovir (GCV). Co-expression of a metabolically related gene in a retroviral vector to potentiate the resistance imparted by a drug resistance gene may be useful for gene therapy for cancer patients.

3T3 Cells↗
PubMed1997

Laryngeal involvement during post kala-azar dermal leishmaniasis in India.

Post kala-azar dermal leishmaniasis (PKDL) involving the mucus membranes is relatively rare on the Indian sub-continent. We describe 3 cases of PKDL presenting with hoarseness of voice. In one case the skin, nasal, oral, oropharyngeal and laryngeal mucosa had nodular and nodulo-ulcerative lesions; in the 2 other cases, genitalia and anorectal mucosa were also affected. Laryngoscopic examination revealed nodular lesions on the vocal cords. Biopsy smear and culture confirmed their leishmanial origin.

Adolescent↗