Sipra Banerjee

Variant forms of DNA polymerase beta in primary lung carcinomas.

DNA polymerase beta (pol beta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. A truncated form of the pol beta protein is expressed in colon and breast cancers. However, the role of the pol beta gene in lung cancer is not known. Thus, we investigated a possible occurrence of pol beta variants in primary lung tumors. The entire cDNA of pol beta obtained by RT-PCR amplification was analyzed for nucleotide sequencing in lung tumor and matched normal lung tissue of the same patient. Three types of variants were detected in squamous, non-small, or large cell carcinomas. The most common variant was a deletion of 87 bp from pol beta cDNA at a site corresponding to exon 11. In addition, a variant exhibiting deletions of 87 and 140 bp together with an insertion of 105 bp was identified in three lung tumors. This is the first report of the occurrence of pol beta variants, possibly splicing variants, in lung cancer. A truncated pol beta protein resulting from variant forms of the gene may impact the function of the enzyme and increase susceptibility to carcinogenesis.

Apoptosis of human carcinoma cells in the presence of potential anti-cancer drugs: III. Treatment of colo-205 and SKBR3 cells with: cis-platin, Tamoxifen, Melphalan, Betulinic acid, L-pdmp, L-PPMP, and GD3 ganglioside

Breast cancer is the most common type of cancer, predominantly among women over 20, whereas colo-rectal cancer occurs in both men and women over the age of 50. Chemotherapy of both cancers affect rapidly growing normal as well as cancer cells. Cancer cells are non-apoptotic. Seven anti-cancer agents (cis-platin, Tamoxifen, Melphalan, Betulinic acid, D-PDMP, L-PPMP, and GD3) have been tested with human breast (SKBR3) and colon (Colo-205) carcinoma cells for their apoptotic effect and found to be positive by several assay systems. Colo-205 cells were obtained from ATCC, and the SKBR3 cells were a gift from the Cleveland Clinic. All of these six agents killed those two cell lines in a dose-dependent manner. In the early apoptotic stage (6 h), these cells showed only a flopping of phosphatidylserine on the outer lamella of the plasma membranes as evidenced by the binding of a novel fluorescent dye PSS-380. After 24 h of the treatment, those apoptotic cells showed damage of the plasma as well as the nuclear membrane as evidenced by binding of propidium iodide to the nuclear DNA. DNA laddering assay viewed further breakdown of DNA by 1% agarose gel electrophoresis analysis. It is concluded that during apoptosis the signaling by Mitochondrial Signaling Pathway (MSP) is stimulated by some of these agents. Caspase 3 was activated with the concomitant appearance of its p17 polypeptide as viewed by Westernblot analyses. Incorporation of radioactivity from [U-14C]-L-serine in total sphingolipid mixture was observed between 2 and 4 micromolar concentrations of most of the agents except cis-platin. However, apoptosis in carcinoma cells in the presence of cis-platin is induced by a caspase 3 activation pathway without any increase in synthesis of ceramide. Published in 2004..

Novel synthetic triterpenoid methyl 25-Hydroxy-3-Oxoolean-12-en-28-Oate induces apoptosis through JNK and p38 MAPK pathways in human breast adenocarcinoma MCF-7 cells

Breast cancer is the most common neoplasm in women and is the leading cause of cancer‐related death for women. Therefore, new agents targeting prevention and treatment of breast cancer are urgently needed. The present study first investigates that a novel triterpenoid Methyl 25‐Hydroxy‐3‐oxoolean‐12‐en‐28‐oate (AMR‐Me) derived from 25‐Hydroxy‐3‐oxoolean‐12‐en‐28‐oic acid (AMR) is a potent inhibitor of cell growth by inducing human breast cancer MCF‐7 cells to undergo apoptosis. AMR‐Me induced DNA fragmentation and PARP degradation which were preceded by changing Bax/Bcl‐2 ratios, cytochrome c release, and subsequent induction of pro‐caspase‐9 and ‐7 processing in breast carcinoma MCF‐7 cells, but it did not act on Fas/Fas ligand pathways and the activation of caspase‐8, suggesting AMR‐Me triggered the mitochondrial apoptotic pathway. The general caspase blocking peptide VAD partially blocked AMR‐Me induced apoptosis. AMR‐Me stimulated p38 mitogen‐activated protein kinase and c‐Jun NH2‐terminal kinase (JNK), but not extracellular signal‐regulated kinase activation during apoptosis. SP600125, a specific inhibitor for JNK and SB203580, a p38 MAPK‐specific inhibitor suppressed AMR‐Me induced apoptosis indicating that activation of JNK and p38 MAPKs involved in the mitochondrial activation‐mediated cell death pathway. Our results suggest that AMR‐Me can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, is critical for both our understanding of cell death events and development of cancer preventive/therapeutic agents.

Defective DNA repair genes in a primary culture of human renal cell carcinoma.

Purpose: Genomic stability is maintained by error-free DNA replication, repair, and recombination. To determine if repair genes contribute to genomic instability, we used a newly established cell line RCC-AJR (from clear-cell renal cell carcinoma) to examine hMSH2 (a mismatch-repair gene) and the gene encoding DNA β polymerase (polβ; a known contributor to base-excision repair). Methods: Coding sequences of hMSH2 and polβ were amplified by the polymerase chain reaction (PCR) using RNA from RCC-AJR cells and matched normal kidney (NK) cells from the same patient. Nucleotide sequences of the PCR products were determined by the dideoxy-DNA method and direct sequencing. Expressions of repair genes were assayed by Western blotting. Microsatellite stability in RCC-AJR cells was assayed by alteration in (CA)n repeats. Results: In the RCC-AJR cells, we detected (a) a deletion of 1476 bp encoding 492 amino acids of hMSH2 cDNA, (b) an 87-bp deletion in the polβ coding sequence, (c) truncated forms of hMSH2 and polβ proteins, and (d) microsatellite instability. Conclusions: This study provides evidence of alterations in hMSH2 and polβ in the homogeneous cell population of an RCC-AJR tumor culture. The data indicate that repair genes may help preserve genomic stability in this cell line. We believe that this new primary RCC-AJR cell line will prove a useful model for investigating the cascade of genetic events in renal cells that leads to renal carcinogenesis.

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of 3H-TdR and 3H/or 14C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 μM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1–4 μM) of these reagents in these cell lines. However, high concentrations (4–20 μM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0–20 μM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer β1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 β1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Lex formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527–35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme. Published in 2004.

Impaired repair activity of a truncated DNA polymerase β protein

Abstract DNA polymerase β (polβ) is an essential enzyme for gap filling synthesis in damaged DNA template involved in base excision repair pathway. A truncated polβ protein is expressed in primary colorectal and breast adenocarcinomas. To determine a possible alteration in the functions of the enzyme, a human cell line named HeLapolβΔ expressing the truncated form of polβ has been established. These cells revealed a significantly reduced level of repair activity evaluated by gap filling synthesis and polβ activity. More importantly, the HeLapolβΔ cells are hypersensitive to MNNG, a DNA alkylating agent. It appears from the responses that the gap filling synthesis of WT cells, a HeLa cell line overexpressing wild-type polβ protein, was inhibited by HeLapolβΔ protein.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, −3, −8, and −9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ‘`Replication Complexes’’ during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-α activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/− aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-α and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 α2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a α2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

A Novel Nuclear Protein, MGC5306 Interacts with DNA Polymerase β and Has a Potential Role in Cellular Phenotype

A novel protein MGC5306 has been identified in yeast–two-hybrid analysis by screening a HeLa cDNA library with a truncated DNA polymeraseβ (polβΔ) as bait. The polβΔ is expressed in various types of cancers. Co-immunoprecipitation–Western blot analysis confirms not only its interaction with polβΔ but also with wild-type polβ. Binding to polβ indicates potential function of MGC5306 in repair pathway. Transfection of cells with MGC5306-GFP and Western blot analysis with anti-MGC5306 antibody reveal its nuclear localization. MGC5306 is expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting MGC5306 is most likely involved in carcinogenesis. An antigrowth activity and modulations of cell cycle events are identified in cells expressing siRNAMGC5306.

Apoptosis of Breast Cancer Cells: Modulation of Genes for Glycoconjugate Biosynthesis and Targeted Drug Delivery

Evidence indicates that blood group-related Lewis (Le) antigens (glycosphingolipids: GSLs and Glycoproteins: GPs) are tumor-associated cell surface molecules. The Lea, Leb, LeX, LeY, and their sialosyl-derivatives, all N-acetylglucosaminyl-containing glycoconjugate antigens, are overexpressed on the surfaces of breast, colon, and ovarian cancer cells during metastasis. A few inhibitors of GSLs (l-/d-PPMP) and DNA (cisplatin) biosynthesis, betulinic acid (a herbal origin of a triterpenoid used for cancer treatment in China), melphalan, and disialosylgangliosides (GD3 and GD1b) induced apoptosis (intrinsic mitochondrial or extrinsic receptor-mediated pathways) in human breast (SKBR-3, MCF-7, and MDA-468) and colon (Colo-205) cells. These chemicals are suggested to be potential anticancer drugs. In this review, we try to compare our recent observations with reported observations from many other laboratories. We discuss the induction of apoptosis in breast and other cancer cells by various new chemicals. We also discuss the biosynthesis and regulation of GSLs in nonapoptotic and apoptotic cancer cells.

Regulation of glycosyltransferase genes in apoptotic breast cancer cells induced by L: -PPMP and cisplatin.

The process of apoptosis is usually triggered by various signals that may have originated extracellularly or intracellularly [1] (Fig. 33.1). The apoptotic signaling initiating from an extracellular source is called an “extrinsic pathway” [2]. The extrinsic apoptotic inducers could be small molecules, such as nitric oxide, hormones such as estrogen [3], or cytokines such as a tumor necrosis factor (TNF-alpha) [4–7]. The death receptor (DR) family plays the most crucial role in transducing the extracellular apoptotic signal to the cytosol apoptotic machinery [8]. The DR family is part of the TNF-receptor superfamily and is usually activated by several cytokines called death ligands. Eight members of the DR family have been identified to date. Although the names of the DRs have varied since the initial discovery of each member, the most common names for each have now been accepted. These are DR1-TNFR1, DR2-CD95, DR3-TRAMP, DR4-TRAILR1, DR5-TRAILR2, DR-6, EDAR, and NGF-R.