Hajnalka Kiss

Human/mouse microcell hybrid based elimination test reduces the putative tumor suppressor region at 3p21.3 to 1.6 cM.

We have previously identified an approximately 7 cM long common eliminated region (CER), involving the 3p21.3 markers AP20R, D3S966, D3S3559, D3S1029, WI‐7947, D3S2354, AFMb362wb9, and D3S32, in human chromosome 3/A9 mouse fibrosarcoma microcell hybrid (MCH) derived SCID mouse tumors. We now report the results of our more detailed analysis on 24 SCID mouse tumors derived from two MCH lines that originally carried intact human chromosomes 3. They were analyzed by fluorescence in situ hybridization (FISH) painting and PCR, using 24 markers covering the region between D3S1611 and D3S1235 at 3p22‐p21.2. D3S32 and D3S2354 were regularly eliminated during in vivo tumor growth, whereas the other 22 markers, D3S1611, ACAA, D3S1260, WI‐692, AP20R, D3S3521, D3S966, D3S1029, D3S643, WI‐2420, MST1, GNAI2, D3S1235, D3S1298, GLB1, WI‐4193, D3S3658, D3S3559, D3S3678, WI‐6400, WI‐7947, and WI‐10865, were regularly retained. We have defined a common eliminated region of approximately 1.6 cM (designated as CER1) inside the 7 cM CER described earlier. CER1 is flanked distally by D3S1029 and proximally by D3S643. Genes Chromosomes Cancer 20:329–336, 1997.

Differential elimination of 3p and retention of 3q segments in human/mouse microcell hybrids during tumor growth

We have previously found that human chromosome 3 was fragmented in the course of in vivo tumor growth of monochromosomal human/mouse (A9 fibrosarcoma parent) microcell hybrids in SCID mice. Marker analysis of tumor cell lines has identified a regularly eliminated 7 cM segment on 3p21.3 referred to as the common eliminated region (CER). The same region is frequently affected by LOH in a variety of human carcinomas. The present study is a comparative chromosome painting, reverse painting, and PCR marker analysis of microcell hybrids (MCHs) that originally contained an intact chromosome 3 from two alternative donors, during and after four passages in SCID mice. We found regular elimination of 3p in parallel with preferential retention of 3q. In addition to CER on 3p, we can now define a common retained region (CRR) on 3q. It includes eight markers between D3S1282 (3q25‐q26) and D3S1265 (3q27‐qter) and spans approximately 43 cM. These observations are concordant with the frequent loss of corresponding 3p regions and the frequent retention, with occasional amplification, of 3q in several types of human tumors. Genes Chromosomes Cancer 20:224–233, 1997.

The transcriptional map of the common eliminated region 1 (C3CER1) in 3p21.3

Occurrence of chromosome 3p deletions in a large number of human tumours suggests the existence of uncharted tumour suppressor gene(s). We previously applied a functional assay, named the Elimination test (Et), for the identification of regions containing tumour growth antagonising genes. This resulted in the definition of chromosome 3 common eliminated region 1 (C3CER1) on 3p21.3, which is regularly eliminated from SCID-derived tumours. Systematic genomic sequencing of 11 PAC clones, combined with comparisons of genomic sequence against EST databases and PCR-based cloning of cDNA sequences allowed us to assemble a comprehensive transcriptional map of 1.4 Mb that includes 19 active genes and three processed pseudogenes. We report four novel genes: FYVE and coiled-coil domain containing 1 (FYCO1), transmembrane protein 7 (TMEM7), leucine-rich repeat-containing 2 (LRRC2) and leucine zipper protein 3 (LUZP3). A striking feature of C3CER1 is a presence of a cluster of eight chemokine receptor genes. Based on a new analysis of the microcell hybrid-derived panel of SCID tumours we also redefined the centromeric border of the C3CER1. It is now located within LRRC2 gene, which is a relative of RSP-1 (Ras Suppressor Protein 1). The detailed knowledge of gene content in C3CER1 is a prerequisite for functional analysis of these genes and understanding of their possible role in tumorigenesis.

Consistent downregulation of human lactoferrin gene, in the common eliminated region 1 on 3p21.3, following tumor growth in severe combined immunodeficient (SCID) mice

Lactoferrin (LF) is one of 19 active genes in the common eliminated region 1 at 3p21.3 identified by us. LF was transfected into mouse fibrosarcoma A9. Fourteen severe combined immunodeficient (SCID) derived tumors from two PI based artificial chromosome (PAC)-transfectants containing the entire LF gene and two LF-cDNA transfectants were analyzed by real time polymerase chain reaction at the DNA and RNA level. Following SCID tumor passage, LF expression was decreased or eclipsed, in all tumors although DNA levels did not change considerably. Promoter methylation and/or rearrangement of the insertion site may be responsible for human LF downregulation in mouse fibrosarcoma derived tumors.

Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3

We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the ≈1.4-Mb common eliminated region 1 (CER1) at 3p21.3. Fluorescence in situ hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR). Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions. Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment). The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure. This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs.

Comparative human/murine sequence analysis of the common eliminated region 1 from human 3p21.3

Interspecies sequence comparison offers an effective approach to identify conserved elements that might have functional importance. We compared 1.32 Mb of C3CER1 (referred also as CER1) from human Chromosome (Chr) 3p21.3 to its orthologous regions on mouse Chr 9F. The corresponding mouse region was found divided into two blocks, but their gene content and gene positions were highly conserved between human and mouse. We observed that two orthologous mouse genes (Xtrp3s1 and Cmkbr1) were duplicated, and this resulted in two additional expressed mouse genes (Xtrp3 and Cmkbr111). We also recognized a large number of conserved elements that were neither exons, CpG islands, nor repeats. We further identified and characterized five novel orthologous mouse genes (Kiaa0028, Xtrp3s1, Fyco1, Tmem7, and Lrrc2).

The microcell hybrid-based “elimination test” identifies a 1-Mb putative tumor-suppressor region at 3p22.2–p22.1 centromeric to the homozygous deletion region detected in lung cancer

We have previously shown that inoculation of human chromosome 3 (chr3)/A9 mouse fibrosarcoma microcell hybrids (MCHs) into severely combined immunodeficient (SCID) mice was followed by the regular elimination of certain 3p regions, whereas a 3q region was retained even after prolonged mouse passage. Using this approach, referred to as the elimination test (Et), we identified a common eliminated region (CER) of about 7 cM at 3p22–p21.3 that was absent in all tumors generated from five MCHs. A second frequently eliminated region (FER, originally called ER2) was found at 3p21.1–p14.2. These segments have been reported to be frequently deleted in a variety of carcinomas. In the following experiments, we have identified at the centromeric border of CER a common eliminated region 1 (CER1) of about 1.6 cM. We now report the results of more detailed analyses of the original tumor panel that contained 30 SCID mouse tumors. Using polymerase chain reaction and chromosome reverse painting, we have identified at the telomeric border of CER a second common eliminated region (designated as CER2). CER2 is flanked distally by RH94338 and proximally by SHGC‐154057. The size of CER2 is about 1 Mb, according to the Homo Sapiens Complete Genome databases at EMBL, and is located about 0.5 Mb centromeric to the known homozygous deletion region, identified in lung cancer. Remarkably, two chemokine‐receptor genes (CCRs), CCR8 and CX3CR1, are located within CER2, whereas seven CCRs were found within CER1.