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Biomedical subjects

D Banerjee

Publications and source records attributed to D Banerjee.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1997

Increased photosensitivity in HL60 cells expressing wild-type p53.

Loss of p53 function has been correlated with decreased sensitivity to chemotherapy and radiation therapy in a variety of human tumors. Comparable analysis of p53 status with sensitivity to oxidative stress induced by photodynamic therapy has not been reported. In the current study we examined photosensitivity in human promyelocytic leukemia HL60 cells exhibiting either wild-type p53, mutated p53 or deleted p53 expression. Experiments were performed using a purpurin, tin ethyl etiopurpurin (SnET2)-, or a porphyrin, Photofrin (PH)-based photosensitizer. Total SnET2 accumulation was comparable in all three cell lines. Uptake of PH was highest in cells expressing wild-type p53 but incubation conditions could be adjusted to achieve equivalent cellular PH levels during experiments that analyzed photosensitivity. Survival measurements demonstrated that HL60 cells expressing wild-type p53 were more sensitive to PH- and SnET2-mediated photosensitization, as well as to UVC irradiation, when compared to HL60 cells exhibiting deleted or mutated p53 phenotypes. A rapid apoptotic response was observed following purpurin- and porphyrin-induced photosensitization in all cell lines. Results of this study indicate that photosensitivity is increased in HL60 cells expressing wild-type p53 and that photosensitizer-mediated oxidative stress can induce apoptosis through a p53-independent mechanism in HL60 cells.

Cell Survival↗
PubMed1997

Prognostic factors for relapse in stage I testicular seminoma treated with surveillance.

PURPOSE: We sought to identify prognostic factors predictive of disease progression in patients with clinical stage I seminoma on surveillance following orchiectomy. MATERIALS AND METHODS: Between January 1981 and December 1993, 201 patients 20 to 86 years old (median age 34) with clinical stage I seminoma were placed on surveillance following orchiectomy. The potential prognostic factors studied included age, tumor size, mitotic count, S phase fraction, ploidy, presence of small vessel invasion, syncytiotrophoblasts and tumor infiltrating lymphocytes, expression of beta-human chorionic gonadotropin and low molecular weight keratin on immunohistochemistry. RESULTS: With a median followup of 6.1 years (range 1.3 to 12.3) 31 patients had relapse for an actuarial 5-year relapse-free rate of 84.9%. The 5-year actuarial survival rate was 97.1% and the cause specific survival rate was 99.5%. On univariate analysis factors predictive of relapse were tumor size (5-year relapse-free rate 88 and 67% for tumors 6 cm. or less and greater than 6 cm., respectively, p = 0.004), age (5-year relapse-free rate 79 and 91% for age 34 years or younger versus older than 34 years, respectively, p = 0.009) and presence of small vessel invasion (5-year relapse-free rate 86 versus 69%, p = 0.01). On multivariate analysis age and tumor size were predictive of relapse, while small vessel invasion approached statistical significance. The risk of relapse in 57 patients with none of the 3 adverse prognostic factors (age greater than 34 years, tumor 6 cm. or smaller and no small vessel invasion) was 6%. CONCLUSIONS: We identified age, size of the primary tumor and small vessel invasion as important prognostic factors for relapse in patients with stage I seminoma treated with surveillance. Further followup and assessment of biological factors are needed to optimize selection of patients at a high risk for relapse who should receive immediate postoperative therapy.

Actuarial Analysis↗
PubMed1997

Haematological changes in buprenorphine-treated mice.

Studies were carried out in adult male Swiss mice to determine whether different haematological parameters like total counts of red and white blood cells, differential counts of white blood cells, haematocrit value and blood haemoglobin level were affected by the synthetic opioid analgesic, buprenorphine, which has currently been known to be abused in several countries by the heroin addicts as a cheap substitute for heroin. The mice were daily given an intraperitoneal injection of buprenorphine (300 micrograms/kg) for 60 consecutive days. A severe leucopenia accompanied by decreases in lymphocyte and monocyte counts, an increase in neutrophil count, a decrease in haematocrit value, a rossette-like adherence of red blood cells to a few neutrophils and platelet satellitism were observed at the advanced stages of treatment. The abnormalities, however, reverted to normal within 45 days following withdrawal of the drug. The necessity of periodic monitoring of the blood picture in human abusers of the drug is suggested.

Agranulocytosis↗
PubMed1996

Characterization of native falcipain, an enzyme involved in Plasmodium falciparum hemoglobin degradation.

In Plasmodium falciparum, a cysteine protease known as falcipain has been implicated in the essential metabolic process of hemoglobin degradation. Parallel lines of investigation, using native or recombinant enzyme, have led to differing conclusions about the specificity and role of this protease. We have now determined that (1) Native falcipain does not cleave hemoglobin unless this substrate has first been denatured by reducing agents, acid-acetone treatment or plasmepsin action. (2) Reducing agents such as glutathione cannot denature hemoglobin in the presence of catalase, which is accumulated in the digestive vacuole. (3) The purified native enzyme has kinetics similar to those obtained with trophozoite extract, but substantially different from those of recombinant enzyme. (4) Although there are numerous cysteine protease genes in the P. falciparum genome, the falcipain gene is the only one whose transcript can be detected in the early intraerythrocytic parasites. We conclude that falcipain likely works by degrading hemoglobin fragments after initial aspartic protease attack has denatured the substrate. We propose that falcipain inhibitors block the initial steps of degradation indirectly by promoting vacuolar accumulation of osmotically active hemoglobin peptides.

Animals↗
PubMed1996

Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST-48 and p90 to 80K-H membrane proteins.

Advanced glycation endproducts (AGEs) are derivatives of nonenzymatic reactions between sugars and protein or lipids, and together with AGE-specific receptors are involved in numerous pathogenic processes associated with aging and hyperglycemia. Two of the known AGE-binding proteins isolated from rat liver membranes, p60 and p90, have been partially sequenced. We now report that the N-terminal sequence of p60 exhibits 95% identity to OST-48, a 48-kDa member of the oligosaccharyltransferase complex found in microsomal membranes, while sequence analysis of p90 revealed 73% and 85% identity to the N-terminal and internal sequences, respectively, of human 80K-H, a 80- to 87-kDa protein substrate for protein kinase C. AGE-ligand and Western analyses of purified oligosaccharyltransferase complex, enriched rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membranes from rat liver or RAW 264.7 macrophages yielded a single protein of approximately 50 kDa recognized by both anti-p60 and anti-OST-48 antibodies, and also exhibited AGE-specific binding. Immunoprecipitated OST-48 from rat rough endoplasmic reticulum fractions exhibited both AGE binding and immunoreactivity to an anti-p60 antibody. Immune IgG raised to recombinant OST-48 and 80K-H inhibited binding of AGE-bovine serum albumin to cell membranes in a dose-dependent manner. Immunostaining and flow cytometry demonstrated the surface expression of OST-48 and 80K-H on numerous cell types and tissues, including mononuclear, endothelial, renal, and brain neuronal and glial cells. We conclude that the AGE receptor components p60 and p90 are identical to OST-48, and 80K-H, respectively, and that they together contribute to the processing of AGEs from extra- and intracellular compartments and in the cellular responses associated with these pathogenic substances.

Amino Acid Sequence↗
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↗