Satya Narayan

ACTIVATION OF ADENOMATOUS POLYPOSIS COLI (APC) GENE EXPRESSION BY THE DNA-ALKYLATING AGENT N-METHYL-N'-NITRO-N-NITROSOGUANIDINE REQUIRES P53

Development of colon cancer is a multistep process frequently involving mutations in both theAPC and p53 tumor suppressor genes. In this study we treated the HCT-116 colon cancer cell line with alkylating agents includingN-methyl-N′-nitro-N-nitrosoguanidine (MNNG),which is known to cause colon cancer in animals, and examined the expression of both APC and p53genes. Exposure of cells with MNNG caused an 8–12-fold increase in the level of APC mRNA and protein. APC induction was shown to result from increased nuclear transcription of the APCgene and correlated with a concomitant increase in the p53 protein level after MNNG treatment. A necessary role for p53 in APCgene regulation is supported by the failure of MNNG to induceAPC expression in cell lines either expressing very low levels of p53 (HeLa cells) or no p53 (K562 erythroleukemia cells). The overexpression of wild-type p53 gene into HCT-116 cells mimicked the effect of MNNG-induced expression of APCmRNA. A direct causal role for p53 in APC gene regulation was further evaluated by transfecting the wild-typep53 gene into K562 cells and observing a 5-fold increase in the APC gene expression. These results support a model featuring a direct link between p53 and APC in response to alkylation-induced DNA damage and suggest a novel role for p53 in a stress-response pathway involving APC.

Activation of the Human DNA Polymerase β Promoter by a DNA-alkylating Agent through Induced Phosphorylation of cAMP Response Element-binding Protein-1

Treatment of cells with the DNA-alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase β (β-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the β-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric β-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the β-pol promoter after treatment of cells with the DNA-alkylating agent MNNG.

Estradiol is trophic for colon cancer in mice: Effect on ornithine decarboxylase and c-myc messenger RNA

The mitogenic role of estradiol on the growth of colon cancer was examined in mice. Sham-operated or ovariectomized mice were injected with cancer cells and received estradiol treatment. Tumor growth was noted: tumor weights were higher in female than male mice. The growth of the tumors was least in ovariectomized mice and highest in estradiol-treated ovariectomized mice. Tumor messenger RNA (mRNA) levels for ornithine decarboxylase (ODC) and proto-oncogenes c-myc, c-fos, and H-ras were examined. Two transcripts (2.2 and 2.7 kilobase pairs) of ODC were observed. The steady-state mRNA levels for ODC paralleled the changes observed in the weight of the tumors in all groups of animals. Less dramatic changes were observed in c-myc mRNA levels. No significant differences were observed in the mRNA levels for H-ras and c-fos. It thus appears likely that an increase in the ODC mRNA levels and, to a lesser extent, an increase in c-myc mRNA levels may be some of the important mechanisms by which estradiol mediates its growth effects on colon cancer cells in vivo.

The presence of a 33-40 KDa gastrin binding protein on human and mouse colon cancer

Human and mouse colon cancers have specific binding sites for gastrin and demonstrate a trophic response to gastrin. In the present study we used radiolabeled gastrin (2-17), to determine the molecular weight of gastrin binding proteins (receptors) on mouse and human colon cancers, by cross-linking methods. Crude membrane aliquots prepared from the tumors were radiolabeled with [125I]gastrin (2-17) +/- 1000 fold excess of unlabeled gastrin and cross-linked with 1 mM disuccinimidyl suberate. The cross-linked radiolabeled binding protein complexes were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The autoradiographs of the gels demonstrated the presence of a predominant band of approximately 33-40 KDa gastrin binding protein, that was specific for gastrin analogs. Our present findings thus indicate that specific gastrin binding proteins/gastrin receptors on colon cancers are primarily present as one band with a molecular mass of approximately 33-40 KDa and are specific for gastrin-like peptides.

Characterization of gastrin binding to colonic mucosal membranes of guinea pigs

Gastrin has significant growth and metabolic effects on colonic mucosal cells. It is, however, not known if gastrin receptors are present on colonic mucosal cells that may directly mediate the reported biological effects of gastrin. In the present studies, the presence of specific gastrin binding sites on colonic mucosal membranes was investigated and the binding sites were further characterized. Crude membranes from colonic mucosa of guinea pigs were analyzed for specific binding to gastrin by our published procedures. A significant number (14.7 ± 1.8 fmoles/mg protein) of high affinity gastrin binding sites (Kd = 0.49 = 0.05 mM) were measured, that were specific for binding gastrin/CCK related peptides and demonstrated negligible binding affinity for all other unrelated peptides examined. In addition a large number of low-affinity (Kd = ∼ μM) binding sites were present. In order to further characterize the molecular size of gastrin binding proteins, we used the chemical cross-linking methods, and observed at least four bands of gastrin binding proteins (GBPs) (∼ 33, 45, 80 and 250 KDa), both under reducing and non-reducing conditions, indicating that these proteins were not sub-units of forms linked by disulfide bonds. Interestingly, majority of the specific gastrin binding sites (∼ 70%) were present on the 45 KDa protein, unlike other target cells of gastrin. The presence of N- and O-linked glycosylated moieties were indicated on the 45 KDa protein, based on enzymatic de-glycosylation studies. The relative binding affinity (RBA) of gastrin and a closely related peptide, cholecystokinin octapeptide (CCK), for GBPs on colonic mucosal membranes was measured in order to determine if GBPs were similar to the CCK-A or CCK-B binding proteins reported in literature. The RBA of gastrin and CCK for displacing the binding of gastrin to the 33, 45, 80 and 250 KDa GBPs on colonic mucosal membranes were calculated to be 39, 100, 78 and 70% (gastrin), and 5.4, 2.9, 3.9 and 2.0% (CCK), respectively, wherein the binding affinity of gastrin for the 45 KDa protein was arbitrarily taken as 100%. Based on the RBA values, it appears more likely that the GBPs on colonic mucosal membranes are more akin to the unique GBPs described on colon cancer cells, and do not represent either the CCK-A or CCK-B binding sites. Based on the cross-linking studies we were not able to determine if the high- and low-affinity binding sites were differentially distributed on the different molecular forms of GBPs measured on the colonic mucosal membranes. The above studies thus indicate for the first time that specific gastrin binding proteins (receptors) are present on colonic mucosal membranes and that these receptor proteins may be directly mediating the observed effects of gastrin on colonic mucosal cells.

IGF-I and IGF-I receptor in mouse colon cancer cells

100 large carrier protein. Although plasma IGF-I is traditionally considered to originate in the hver (1), recent reports indicate that it is synthesized at multiple sites in the body (2,3). Preliminary studies also indicate that IGF-I receptors are widely distributed in the GI tract of humans and rats (4), suggesting that IGF-I is likely to be an important regulator of growth and metabolism of the normal cells

High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 ±0.02 nM) for BBS were measured, with a binding capacity of 27.3 + 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyro-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14–27) = GRP (18–27) > GRP (1–27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-ψ-(CH2NH) Lcu14]-BBS (LL-BBS) and D-Phe6, BN(6–13) propylamide (D-Phe6,BN(6–13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70–80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.