Identification of the common eliminated region (CER1) by the microcell hybrid based 'elimination test'

Abstract

The aim of elimination test (Et) is to identify tumor antagonizing genes from chromosome regions that are regularly deleted in microcell hybrid (MCH) derived tumors through serial SCID mice passages. Et so far was focused on the short arm of human chromosome 3 (chr 3) due to its frequent deletions in 21 tumor types including renal cell carcinoma (RCC), small cell lung carcinoma (SCLC) and breast cancer (BRC). A common eliminated region (CER) on 3p21.3 was identified in tumor panels by PCR marker and FISH analysis and was narrowed down successively from 40 cM to 7 cM.

In the present thesis, CER was further reduced to 1.6 cM segment designated as CER1 in the human / mouse MCH system (chr 3 / mouse fibrosarcoma line A9). It contained 2 markers D3S2354 and D3S32 and located at the centromeric part of the former CER. For initiation of positional cloning of genes, CEN1 was filled with 12 additional markers by using extra tumors and subsequently covered by a PAC contig. The measurement of the contig was done by fiber-FISH and the size of CER1 was estimated to ~1 Mb. During the course of coverage of CER1, we found that the cluster of chemokine receptor genes CCR1, CCR3, CCR2, CCR5 and CCR6 and lactoferrin gene (LF) with reported tumor inhibitory activity have been mapped to this region.

In order to test the validity of Et results obtained from the human / mouse MCH system, we initiated a human / human Et by establishing new chr 3 / human tumor MCHs. A9Hytk3 (a microcell hybrid that contains a single cytogenetically intact human chr 3 on mouse fibrosarcoma A9 background) and nonpapillary renal cell carcinoma line KH39 (uniparental-disomy) were used as donor and recipient. Four MCHs were generated and designated as YYK1, 2, 3 and 4. Twenty derived tumors were analysed by chr 3 arm specific painting, 27 polymorphic and 19 FISH markers. The results showed that tumors derived from YYK2, 3 and 4, which maintained the intact exogenous chr 3 in vitro, have lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, 4 tumors contained mixed cell populations, that lacked either the exogenous or one endogenous 3p derived from KH39. While tumors from YYK1, which showed deletions within CER1 already on the exogenous chr 3 in vitro, remained essentially unchanged in 8/9 derived tumors. We could conclude that the human / human Et identifies similar eliminated regions on chr 3 as the human / mouse Et, and the elimination of CER1 could be regularly associated with tumor growth.

In the course of large scale sequencing of CER1 PAC-contig, two overlapping PACs (86:16E and RP5- 965c9) from the centromeric part of CER1 were sequenced first. Using the sequences of 86:16E and RP5- 965c9 as a query for BLAST database search revealed two genes. The last two exons of leucyl-tRNA synthetase (KIAA0028) was located in the telomeric part of PAC 86:16E, the 5 end and the first exon of the second gene was towards to the centromere of 86:16E. Multiple human and mouse ESTs that were corresponding to the second gene were also found by the BLAST search. For assembling the full cDNA and filling the gaps, primers were designed for amplifying the Marathon placental cDNA library, followed by sequencing of the PCR products. BLAST analysis of cDNA and predicted protein sequence revealed striking similarity with other members of the LIM domaincontaining genes. This gene was therefore named LIMD1. The open reading frame of this gene is 2028 bp and its Exon 2-8 contains 3 tandemly ordered LIM domains. LIM domains contain a cysteine-rich consensus sequence with two distinct zinc-binding subdomains, which mediate protein-protein interactions.

Human lactoferrin (LF) gene - reported earlier for tumor and metastasis inhibitory function - is one of the 20 genes located in the common eliminated region 1 (CER1) at 3p21.3. We transfected a PAC containing the entire LF gene and its promoter, and a LF-cDNA into the mouse fibrosarcoma cell line A9. Fourteen SCID derived tumors from two independent PAC and two cDNA transfectants were analysed by real time PCR at DNA and RNA level. All of them had lost or diminished LF expression, in contrast with the relatively constant DNA levels. A remarcable diversification of the insertion sites has been occurred in vivo, during the SCID tumor development. Two tumors that showed strong downregulation were chosen for methylation analysis by bisulfite treatment and sequencing. Six CpG sites out of 14 in the LF promoter have been methylated in both tumors. No methylation was found in the in vitro transfectants. Epigenetic inactivation of LF promoter may be responsible for its downregulation upon tumor growth.