Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.

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We assessed the feasibility of clonality analysis with human androgen receptor gene polymerase chain reaction in terms of the sensitivity and specificity for normal and cancerous colonic tissues taken from fourteen informative cases selected from 22 women with colonic adenocarcinoma. Ten crypts microdissected from each 10- Òm-thick cryostat sections and whole tissues were used as samples. DNA was extracted from the samples and amplified with and without prior enzyme digestion. These products were analyzed by capillary electrophoresis for clonality. Of the whole-tissue DNA, none of the normal tissues and seven (50.0%) of the cancerous tissues showed monoclonality. Of the microdissected samples, monoclonality was found in 88.4% (107/121) of normal crypts and 95.9% (117/122) of cancerous crypts. Samples composed of crypts with short and long alleles were found in eight of the 14 normal colonic mucosae, but in none of the cancerous tissues. We concluded that the sensitivity of this method is limited for both whole-tissue DNA and microdissected-tissue DNA, because monoclonality from small samples does not always indicate monoclonality of the entire lesion. The high specificity of this method, however, allows polyclonal results in whole tissues to be confirmed by additional analysis of microdissected tissues.

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