SUMMARY:The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16 INK4a and p14 ARF , whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14 ARF and p16 INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14 ARF and p16 INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1␤ of p14 ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14 ARF , documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14 ARF and p16 INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14 ARF was inversely correlated with p53 overexpression (p ϭ 0.02). p14 ARF and p16 INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p ϭ 0.02). Methylation of the p14 ARF gene occurred more frequently in well-differentiated adenocarcinomas (p ϭ 0.005), whereas the p16 INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p ϭ 0.002). The present results underline the role of p14 ARF and p16 INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14 ARF and p16 INK4a , might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways. (Lab Invest 2001, 81:217-229).

Transglutaminases (EC.2.3.2.13, protein-glutamine: amine ␥-glutamyl-transferase) are a superfamily of enzymes which catalyze the formation of intra-and intermolecular ␥-glutamyl-

We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, a1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1a,25-dihydroxyvitamin D 3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering.