Investigation in Liver Tissues and Cell Lines of the Transcription of 13 Genes Mapping to the 16q24 Region That Are Frequently Deleted in Hepatocellular Carcinoma Philippe Riou, Raphae ¨l Saffroy, Je ´rome Comoy, Marine Gross-Goupil, Jean-Paul Thie ´ry, Jean-Franc ¸ois Emile, Daniel Azoulay, Dominique Piatier-Tonneau, Antoinette Lemoine, and Brigitte Debuire 1 Service de Biochimie et Biologie mole ´culaire [P. R., R. S., J. C., M. G-G., J-P. T., A. L., B. D.], Service d’Anatomie pathologique [J-F.E.], and Centre He ´patobiliaire Ho ˆpital Universitaire Paul Brousse [D. A.], UPRES 1596-Faculte ´ de Me ´decine Paris-Sud, 94804 Villejuif Cedex, France, and Ge ´ne ´tique Mole ´culaire et Biologie du De ´veloppement, FRE 2376, Centre National de la Recherche Scientifique, 94801 Villejuif Cedex, France [D. P-T.] ABSTRACT Many studies have associated chromosomal deletions in the 16q24 region with human cancers, including hepatocel- lular carcinoma. A more limited region around the micro- satellite D16S402 has been shown implicated in the meta- static spread of hepatocellular carcinoma, prostate cancer, and Wilms’ tumors. It is likely that one or more tumor suppressor genes are located in this 16q24 area. We used SYBR Green reagents to quantify, by real- time quantitative RT-PCR, the production of mRNA for 13 genes mapping to 16q24. The locations of these genes were determined from published human genome sequencing data. We studied mRNA levels in 10 liver tumor tissues, 10 non- tumor liver tissues, five hepatoma cell lines, and in isolated hepatocytes. Results were compared with those for loss of heterozygosity observed in the D16S402 region and recur- rence. No down-regulation was observed in tumor tissues. Two genes were consistently overexpressed: OKL38 and CDH13. CDH13, which functions in cell-cell adhesion, seems to be involved in liver carcinogenesis. However, no relation- ship was observed between the expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested seemed to be a good candidate for a major tumor suppressor gene in liver carcinogenesis. Our results suggest that additional unknown genes in- volved in carcinogenesis are located in the 16q24 area. INTRODUCTION HCC 2 is one of the most frequent human cancers world- wide (1). However, the molecular mechanisms underlying HCC tumorigenesis and tumor metastasis are still poorly understood. HCC, like other solid tumors, seems to develop following mul- tiple genetic events (2), including the functional inactivation of TSGs and the activation of oncogenes. Previous studies of LOH have suggested that several genomic regions may be involved in liver carcinogenesis. These regions are mostly located on chro- mosome arms 1p, 4q, 5q, 6q, 8p, 9p, 11p, 13q, 16p, 16q, and 17p, indicating that dysfunctions of diverse tumor or metastasis suppressor genes located on these chromosomes are involved in the development of HCC (3–5). LOH on the long arm of chromosome 16 has been reported to be frequent in several human cancers, including HCC. Anal- yses of LOH frequencies (6 –12), comparative genomic hybrid- ization (13–15), and aberrant DNA methylation (16, 17) have suggested that one or more TSGs involved in the development of HCC map to chromosome 16q. LOH on chromosome 16q has been correlated with clinicopathological characteristics, such as the degree of differentiation, size, and the occurrence of metas- tases, indicating that LOH on chromosome 16 may be involved in the progression of HCC (6, 12). Subsequent molecular analyses in HCC (5, 10 –12, 15, 18 –20), prostate (21, 22), ovarian (23–25), breast (26, 27), bladder (28), and Wilms’ (29 –32) tumors have identified the 16q24 region as a major region of LOH, associated with meta- static and aggressive behavior of the cancer (21, 22, 29, 32–34). Mashimo et al. (33) recently used microcell-mediated chromo- some transfer into a highly metastatic rat prostatic cancer cell line to show that microsatellite marker D16S402 was retained in microcell hybrids displaying significant reduction in lung me- tastasis. We have shown that changes in the D16S402 micro- satellite are frequent in European HCC patients and are corre- lated with a higher risk of recurrence (35). LOH studies in several other types of tumor have also suggested that there is at least one TSG near the D16S402 locus (19, 23, 26, 31, 36). Data from the human genome sequencing program were recently published (37), and information concerning the fine mapping of genes is now available on via the internet. 3 We identified 13 genes mapping to the 16q23.3–24.1 chromosomal region between the D16S422 and D16S3037 loci, encompassing a region of approximately 2 cM, including D16S402 (Fig. 1). We selected only known genes and full-length mRNAs corre- Received 10/19/01; revised 4/22/02; accepted 6/4/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Service de Biochimie et Biologie mole ´culaire, Ho ˆpital Universitaire Paul Brousse, UPRES 1596-Faculte ´ de Me ´decine Paris-Sud, 14 avenue Paul Vaillant Couturier, 94804 Villejuif Cedex, France. 2 The abbreviations used are: HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; TSG, tumor suppressor gene; NL, normal liver RNA; NTL, nontumor liver RNA; TL, tumoral liver RNA. 3 Internet address: www.genome.cse.ucsc.edu. 3178 Vol. 8, 3178 –3186, October 2002 Clinical Cancer Research