Induction of Apoptosis in Metastatic Breast Cancer Cells: XV. Downregulation of DNA Polymerase-α - Helicase Complex (Replisomes) and Glyco-Genes.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2 h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2 h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24 h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48 h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

Association between newly identified variant form of DNA polymerase beta (Δ 208-304) and ovarian cancer.

BACKGROUND: Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polß), which removes the deoxyribose phosphate group (dRP) and fills in the gap with a nucleotide after the DNA lesion is excised. It has been shown that more than thirty percent of breast, bladder, esophageal, colon, and gastric cancer samples studied so far have exhibited DNA polymerase beta mutation. AIM: To examine the association between polß polymorphism and ovarian cancer, case control study was performed using one hundred fifty two cancer samples and non-metastatic normal samples from the same patients in Indian population. DESIGN: The polß polymorphism was studied in ovarian carcinoma tissues samples initially by RT-PCR followed by sequencing and then by western blot analysis. RESULT: A new type of variant was detected along with the WT allele (polßΔ _{208-304}). Stage IV samples have shown a significant factor for cancer progression in ovarian cancer patients of India [OR=3.58; 95% CI (1.6-7.9); and p=0.001]. The association study involving serous type and the variant showed a tendency towards ovarian carcinogenesis [OR=1.57; 95% CI (0.8-3.1); p=0.19]. The western blot analysis result indicates that the specific deletion appears to be associated with disease progression. CONCLUSION: The result reveals that this variant form of polß is a predisposing factor for stage IV ovarian cancer samples in Indian population.

Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration.

The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet the molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Over-expression of PIAS3 effectively changes cell shape and inhibits GBM cell migration. We focused on the molecular target(s) of PIAS3 stimulated sumoylation, which play an important role in the inhibition of GBM cell motility. Here we report, through the immunoprecipitation with SUMO1 antibody, followed by proteomic analysis, the identification of vimentin (vimentin354), a nuclear component in GBM cells, as the main target of sumoylation promoted by PIAS3.

Novel semisynthetic triterpenoid AMR-Me inhibits telomerase activity in human leukemic CEM cells and exhibits in vivo antitumor activity against Dalton's lymphoma ascites tumor.

Telomerase, a ribonucleoprotein complex of hTERT and hTER, has been reported to be associated with carcinogenesis and multidrug resistance (MDR). Methyl-25-hydroxy-3-oxoolean-12-en-28-oate (AMR-Me) is a novel semisynthetic triterpenoid, derived from a triterpene acid isolated from the stem bark of a tropical tree Amoora rohituka grown wild in India. We examined the role of telomerase in mediating the growth suppression of human acute lymphoblastic leukemic CEM cells by AMR-Me. The results showed that AMR-Me inhibited the growth and viability of CEM cells, induced apoptosis and cell cycle arrest in G(2)+M phase. AMR-Me treatment resulted in suppression of hTERT expression and a concomitant inhibition of telomerase activity. The in vivo antitumor activity of AMR-Me was determined using mice inoculated with Dalton's lymphoma ascites tumor cells. Intraperitoneal administration of the AMR-Me at doses of 1 or 3mg/kg, increased the survival rate by 121% and 133% respectively, without weight change over the treatment period. Our results suggest that AMR-Me inhibits telomerase activity by decreasing the hTERT expression and induces apoptosis in human lymphoblastic leukemic CEM cells, thus providing the molecular basis for the development of AMR-Me as a novel chemotherapeutic agent against leukemia.

Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with L-PPMP.

Functions of glycosphingolipids on the eukaryotic cell membranes during the onset of oncogenic processes and cell death are not well understood. Inhibitors of glycosphingolipid biosynthesis were recently found to trigger apoptosis in many carcinoma including breast cancer SKBR-3, MCF-7, and MDA-468 cells through either intrinsic or extrinsic apoptotic pathways as we previously reported. These anti-cancer inhibitors could increase ceramide concentration by blocking functions of glycolipid glycosyltransferases (GLTs). In this study, using a novel fluorescent dye (ASK-0) revealed the damage of cell organelle membranes by an inhibitor of glucosylceramide biosynthesis (L-PPMP). A highly drug- and cell-dependent regulation of MAPKs was also found by cis-platin and L-PPMP when inducing apoptosis in SKBR-3, MCF-7, and MDA-468 cells. A dose and time-dependent regulation of GLTs were investigated by enzymatic assay and DNA microarray analyses. These GLTs are involved in biosynthesis of Le(X) and sialosyl-Le(X) (neolactosyl-ceramide series) such as GalT-4 (UDP-Gal: LcOse3cer beta-galactosyltransferase, GalT-5 (UDP-Gal: nLcOse4Cer alpha1, 3galactosyltransferase, FucT-3 (GDP-Fucose: LM1 alpha1, 4fucosyltransferase). A similar effect was observed with the GLTs involved in the biosyntheses of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: GgOse4Cer alpha2, 3sialyltransferase, and SAT-2 (CMP-NeuAc: GM3 alpha2, 8sialyltransferase). The glycol-related gene DNA-microarrays also suggested the transcriptional regulation of several GLTs involved in the biosynthesis of neolactosylceramide containing cell-surface antigens in these apoptotic breast carcinoma cells. In the early apoptotic stages (2 to 6 h after L-PPMP treatment) in addition to GlcT-1 gene, several genes (betaGalTs and betaGlcNAcTs) in the SA-Le(a) pathway were stimulated.

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.

Mammary carcinogenesis in transgenic mice expressing a dominant-negative mutant of DNA polymerase beta in their mammary glands.

DNA polymerase beta (polbeta) is a major contributor to mammalian DNA damage repair through its gap-filling DNA synthesis and 5'-deoxyribose phosphate lyase activities. In this way, polbeta plays pivotal roles in the repair of oxidative DNA damage, replication, embryonic survival, neuronal development, meiosis, apoptosis and telomere function. A 36 kDa truncated polbetaDelta protein is expressed in human colorectal, breast, lung and renal carcinomas, but not in normal matched tissues. Interestingly, a binary protein-protein complex of polbetaDelta and X-ray cross-complementing group 1 acts as dominant-negative mutant. In this study, the potential tumorigenic activity of polbetaDelta was examined in nude and transgenic mouse models. Mouse embryonic fibroblasts (MEFs) expressing polbetaDelta in the absence of endogenous polbeta exhibited increased susceptibility to N-methyl-N-nitrosourea (MNU)-induced morphological transformation as compared with cells expressing wild-type (WT) polbeta. This was accompanied by reduced gap-filling DNA synthesis activity. Anchorage-independent transformed cells derived from polbetaDelta-expressing MEFs induced 100% tumor occurrence in nude mice. To support these data, we established transgenic mice expressing polbetaDelta specifically in the mammary glands from a whey acidic protein promoter-driven transgene. This is the first report of transgenic mice with tissue-specific expression of polbetaDelta. MNU-induced tumor formation was analyzed in transgenic mice expressing polbetaDelta together with endogenous WT polbeta in their mammary glands and in normal control mice expressing only WT polbeta. The latent period of tumor appearance was markedly shorter and tumor incidence was significantly higher in transgenic animals than in control animals treated under the same conditions. These results indicate that cells expressing the mutant polbetaDelta display an enhanced sensitivity to MNU that probably underlies an increased susceptibility to tumorigenesis.

Novel triterpenoid 25-hydroxy-3-oxoolean-12-en-28-oic acid induces growth arrest and apoptosis in breast cancer cells.

25-Hydroxy-3-oxoolean-12-en-28-oic acid (Amooranin-AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A herbal preparation used for the treatment of cancer by the Ayurvedic system of medicine contains the stem bark of Amoora rohituka as one of the ingredients. In this paper, we show that AMR displays a strong inhibitory effect on survival of human breast carcinoma MDA-468, breast adenocarcinoma MCF-7 cells compared to breast epithelial MCF-10A control cells. A 50% decrease in cells (IC50) ranged from 1.8 to 14.6 microM and cell growth was suppressed by arresting cell cycle at G2 + M phase. AMR effectively induces apoptosis and triggered a series of effects associated with apoptosis including cleavage of caspase-8, -9, -3, Bid and ER stress in MDA-468 cells and caspase- 8, -9, -6 and Bid in MCF-7 cells, release of cytochrome c from the mitochondria, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation with a concomitant upregulation of p53, Bax and down-regulation of Bcl-2 in MDA-468 cells, but Bax unchanged in MCF-7 cells. The use of caspase blocking peptides and acridine orange staining confirmed the involvement of primarily caspase-9 and -3 in MDA-468 cells with mutated p53 and primarily caspase-8, -9 and -6 in MCF-7 cells expressing wt p53. We also observed in MCF-7/p53siRNA cells AMR treatment caused reduced expression of Bcl-2 without affecting levels of Bax similar to MCF-7 cells treated with AMR and proteolytic activation of Bax in MDA-468 cells. These results suggest that AMR induces apoptosis in human breast carcinoma cells via caspase activation pathway and likely it is a p53-independent apoptosis.

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-alpha 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-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 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 beta 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 polymerasebeta (polbetaDelta) as bait. The polbetaDelta is expressed in various types of cancers. Co-immunoprecipitation-Western blot analysis confirms not only its interaction with polbetaDelta but also with wild-type polbeta. Binding to polbeta 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 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-(14)C]-L-serine in total sphingolipid mixture was observed between 2 and 4 micromolar concentrations of most of the agents except ci s-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.

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.

Differential PIAS3 expression in human malignancy.

STAT3 mediated signaling pathways can be inhibited by PIAS3 (protein inhibitor of activated STAT3), which was recently found to regulate protein stability and function by its SUMO (small-ubiquitin like modifiers) ligase activity in promoting sumoylation of important nuclear proteins. Over-expression of PIAS3 can induce apoptosis in prostate cancer cell lines both in vitro and in vivo. Checking endogenous PIAS3 expression in various human specimens by immunohistochemistry, we report that basal amounts of PIAS3 exist in the nucleus in a majority of epithelial and endothelial cells. Increased PIAS3 expression was observed in 100/103 samples examined in a variety of human cancers including lung, breast, prostate, colon-rectum, and brain tumors. Differential PIAS3 expression and the specific patterns may be useful as a molecular marker of human cancer.

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 (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) 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 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) 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.

Alterations of transforming growth factor beta receptor II, insulin growth factor receptor II genes in microsatellite unstable prostate carcinomas.

DNA from 45 primary prostate tumors and corresponding normal tissues were analyzed to detect whether the alterations of transforming growth factor beta receptor II (TGFbetaRII) and insulin growth factor receptor II (IGFRII) are associated with microsatellite instability (MSI). We identified that 25 tumors were microsatellite unstable (55%). The remaining 20 tumors are found to be microsatellite stable. Loss of heterozygosity (LOH) was also tested at various loci. Results indicate that in case of TGFbetaRII, the rate of frame-shift mutation depends on the number of polyadenine [poly(A)] tracts. Twelve percent of the tumors had frame-shift alteration at BAT-RII locus which has 10 poly(A) repeats. Twenty percent of the tumors had frame-shift at BAT-25 locus which has 25 poly(A) repeats. In addition, IGFRII gene was examined for the presence of mutation in the repetitive sequences. Seven of the 25 tumors showed deletion of a G within eight poly(G) repeats. Besides these changes there were two tumors which showed a novel insertion of A within this poly(G) repeat making a change in 9 samples (R4, 36%). On the other hand, 4 tumors showed changes within the 5CT repeats. In addition, 3 tumors showed another novel insertion of C within the CT repeats.