A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.

We have determined the nucleotide sequence of two different mouse alpha-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively are the major alpha-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5' noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict alpha-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland alpha-amylase mRNA is not initiated at the AUG codon nearest the 5' terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.

Abstractp53, a major sensor of DNA damage, is a transcription factor that, depending on its phosphorylation status, regulates the cell cycle, DNA repair, or apoptosis. The protein kinase C (PKC) family of isozymes is also implicated in cell cycle and programmed cell death (PCD) control and has recently been shown to influence p53 function. Using three human colon adenocarcinoma cell lines SW480, EB-1, and HCT116 that either lack p53 function and were engineered to express inducible wild-type p53 (wt p53), or that constitutively express wt p53, we show that phorbol ester-mediated PKC activation potentiates p53-induced PCD. Despite the effectiveness of PKC/p53 synergy in inducing SW480 tumor cell death, however, a fraction of the cells invariably survive. To address the putative mechanisms that underlie resistance to PKC/p53-induced cell death, we generated a phorbol 12-myristate 13-acetate/p53-resistant SW480 subline and compared the gene expression profile of resistant and parental cells by DNA microarray analysis. The results of these experiments show that PKC/p53-resistant cells express a higher level of several matrix metalloproteinases (MMP), including MMP-9, MMP-10, and MMP-12, and corresponding real-time PCR assays indicate that p53 is a negative regulator of MMP-9 gene expression. Using MMP inhibitors and MMP-specific small interfering RNA, we show that MMP function confers protection from PKC/p53-induced apoptosis and identify the protective MMPs as MMP-9 and MMP-10. Taken together, these observations provide evidence that MMPs are implicated in tumor cell resistance to the synergistic proapoptotic effect of PKC and p53. (Cancer Res 2005; 65(10): 4261-72)

The sequence of 1,773-nucleotide major and 1,806-nucleotide minor mouse liver alpha-amylase mRNAs differ only with respect to approximately 30 additional residues at the extreme 5' terminus of the minor species. Comparison of the liver alpha-amylase mRNAs with their salivary gland counterpart reveals that these mRNAs share identical coding and 3'-noncoding sequences, but contain distinct 5'-terminal residues. These data suggest that all three mRNAs might be transcribed from the same gene.

No abstract

The effects of functionalized aryl beta-D-glycopyranosides (glycomers) on the proliferation, survival, and apoptosis of human glioblastoma cells in culture were evaluated as a way to control tumor progression. The results showed that inhibition of growth and/or induction of apoptosis can be achieved by these molecules in human glioblastoma cells. Inhibition of DNA synthesis precedes induction of apoptosis and growth inhibition. The substituents at C-1, C-2, C-3,C-4, and C-6 on the pyranosidic scaffold are important to modulate the action and the efficacy of these molecules. Human fibroblasts and brain-derived endothelial cells were less sensitive to glycomers than tumor cells. Thus, functionalized aryl beta-D-glycopyranosides represent a new class of molecules potentially able to control the progression of brain tumors.

Endothelin (ET)-1 can act as an autocrine/paracrine growth factor or an antiapoptotic factor in human cancers. To study the role of ET-1 in human colon cancer, proliferation and apoptosis of colon carcinoma cells was investigated using human HT-29 and SW480 colon carcinoma cells. ET-1 was secreted by these cells. Treatment of cells with bosentan, a dual ET A/B -receptor antagonist, decreased cell number. Inhibition of DNA synthesis by bosentan was observed only in the presence of serum. Exogenously added ET-1 did not increase DNA synthesis in serum-deprived cells. SW480 cells were sensitive and HT-29 cells were resistant to FasLinduced apoptosis. Bosentan sensitised resistant HT-29 cells to FasL-induced, caspase-mediated apoptosis, but not to TNF-a-induced apoptosis. Bosentan and/or FasLigand (FasL) did not modulate the expression of caspase-8 or FLIP. Bosentan sensitisation to apoptosis was reversed by low concentrations (10 À13 -10 À10 M), but not by high concentrations (10 À9 -10 À7 M) of ET-1. These results suggest that the binding of ET-1 to high-affinity sites inhibits FasL-induced apoptosis, while the binding of either ET-1 or receptor antagonists to low-affinity sites promotes FasL-induced apoptosis. In conclusion, endothelin signalling pathways do not induce human colon cancer cell proliferation, but are survival signals controling resistance to apoptosis.

No abstract