LOH studies show that genetic alterations of chromosome 9p occur in a variety of tumor types including lung cancer, suggesting the presence of TSGs critical in the development of human cancers. Localization of HDs in tumor DNA is an important strategy to identify potential TSGs. A detailed deletion mapping study with 55 integrated STSs, microsatellite markers, and four known genes located on chromosome 9p21–24 in human lung cancer cell lines led to the identification of five regions of HDs. Of these deletions, two frequently deleted regions are clustered at the TSG p16/CDKN2A locus and the previously identified deletion at the D9S126 locus. These findings are consistent with previous reports that the p16/CDKN2A locus is a primary target for deletion in 9p. Nevertheless, additional deletions and TSGs appear to exist on chromosome 9p (Wiest et al., 1997
). Interestingly, three independent novel HDs, distal to, but not contiguous with, the deletion of p16/CDKN2A, were identified in two NSCLC cell lines, NE-18 and Nu 6-1. Since the STSs and microsatellite markers used in this study are all available in the genome sequence database, these findings will provide a convenient method to analyse the genes and genome for the deleted regions described in this report. Results will be necessary and helpful to understand the involvement of HD in human carcinogenesis and how the genome rearranged chromosome 9p in the cell lines bearing the HDs.
As reported previously, both NE-18 and Nu 6-1 were clones of the human squamous lung carcinoma tumor RG-SLC-L11 (Stranahan et al., 1996
). Therefore, it is not surprising to see the identical deletion patterns revealed by PCR, quantitative real-time PCR and Southern blot in these two cell lines. However, the mechanisms for the HDs in these cell lines remain to be explored. It will be useful to screen additional cell lines and primary tumors to determine if these novel deletions occur frequently. In addition, molecular cytogenetic studies such as SKY and FISH with probes from deleted regions in both cell lines may provide a clue to the cause of the chromosomal rearrangements.
The region containing D9S126 is frequently reported to be affected by HD or LOH in human cancers, for example, breast cancer, squamous cell lung cancer, melanoma, hepatocellular, suggesting that the region may contain potential TSGs. One intronless gene (TUSC1) in this region and its mouse homologue (Tusc1) were characterized in detail in this report. Two major TUSC1 transcripts, TUSC1-L and TUC1-S, were detected in a wide range of adult tissues by Northern blot analysis, indicating a ubiquitous gene expression pattern even though their expression levels differed (). Similar expression patterns for Tusc1 were also revealed in mouse tissues (). In addition, one transcript of approximately 1.1 kb was only present in human testis, and the corresponding cDNA remains to be elucidated. HD of TUSC1 in genomic DNA from the cell lines Nu 6-1, NE-18, and H290 were clearly demonstrated by multiplex PCR and in Southern blots (). However, further studies are required to determine if the weak 8 kb band detected with the TUSC1 probe represents a gene homologue of TUSC1.
RT–PCR for p16/CDKN2A and TUSC1 with cDNA from the lung tumor cell lines indicated the absence of p16/CDKN2A and TUSC1 transcripts in the cell lines with HD. It should be noted that cell line H460 lacks the expression of TUSC1 without harboring a HD. In addition, three cell lines with reduced expression of TUSC1 do not harbor an HD of the TUSC1 locus. Therefore, expression inactivation may be caused by other mechanisms, such as hypermethylation of the CpG island in the promoter region (Jones and Laird, 1999
; Baylin et al., 1998
; Baylin and Herman, 2000
; Jones and Baylin, 2002
). Finally, expression levels of TUSC1-L are reduced in two cell lines, H358 and SHP77, without apparent changes in p16/CDKN2A expression levels, suggesting that expression from these two loci are not coordinated.
Taken together, we provided strong evidence for other regions of HD on chromosome 9p in addition to the p16/CDKN2A locus in human lung cancer cells. These regions are likely to harbor potential TSGs important for the development of lung carcinogenesis. TUSC1, a novel intronless gene, was not expressed or showed reduced expression in a subset of lung cancer cells, and these findings suggest that TUSC1 may play a role as a tumor suppressor in lung tumorigenesis. Further functional and biological studies are expected to confirm if TUSC1 is a TSG and enable us to better understand the molecular mechanism of human lung cancer progression.