Stefan Imreh

Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma.

We have recently developed an allele titration assay (ATA) to assess the sensitivity and influence of normal cell admixture in loss of heterozygosity (LOH) studies based on CA-repeat. The assay showed that these studies are biased by the size-dependent differential sensitivity of allele detection. Based on these data, we have set up new criteria for evaluation of LOH. By combining these new rules with comparative genome hybridization (CGH) we have shown the presence of interstitial deletions in renal cell carcinoma (RCC) biopsies and cell lines. At least three out of 11 analysed RCC cell lines and three out of 37 biopsies contain interstitial deletions on chromosome 3. Our study suggests the presence of several regions on human chromosome 3 that might contribute to tumor development by their loss: (i) 3p25-p26, around the VHL gene (D3S1317); (ii) 3p21.3-p22 (between D3S1260 and D3S1611); (iii) 3p21.2 (around D3S1235 and D3S1289); (iv) 3p13-p14 (around D3S1312 and D3S1285). For the first time, AP20 region (3p21.3-p22) was carefully tested for LOH in RCC. It was found that the AP20 region is the most frequently affected area. Our data also suggest that another tumor suppressor gene is located near the VHL gene in 3p25-p26.

Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

B cell phenotype-dependent expression of the Epstein-Barr virus nuclear antigens EBNA-2 to EBNA-6: studies with somatic cell hybrids.

The expression of the transformation-associated Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) 1 to 6 and membrane protein LMP-1 was studied in a series of somatic cell hybrids derived from the fusion of the EBV-transformed lymphoblastoid cell line (LCL) KR-4, and the EBV-carrying Burkitts lymphoma lines Daudi, P3HR-1 and Raji, with non-B cell lines of fibroblast, erythroid, myeloid and epithelial origin. Expression of EBNAs 2 to 6 was down-regulated in the hybrids in parallel with extinction of the B cell markers CD19, CD20, CD21, CD23, HLA class II, and surface or cytoplasmic immunoglobulin. LMP-1 was expressed independently of EBNA-2 in hybrids derived by the fusion of the LMP-1-positive KR-4 and P3HR-1 cell lines with epithelial and myeloid cells, respectively. LMP-1 was down-regulated in hybrids derived by the fusion of P3HR-1 with an erythroid cell line and in the hybrid between Raji and a mouse fibrosarcoma line. EBNA-1 was the only EBV antigen that was regularly expressed in the hybrids regardless of the dominating cellular phenotype. The autonomous expression of EBNA-1 suggests that its regulatory pathway is independent of phenotype-associated cellular or viral factors. In contrast, the expression of EBNAs 2 to 6 appears to require a B cell environment. EBNA-2 was shown to contribute to the regulation of LMP expression in B cells. We show that in LCL-carcinoma hybrids the dominating epithelial phenotype is permissive for LMP expression in the absence of EBNA-2.

Mitotic Infidelity and Centrosome Duplication Errors in Cells Overexpressing Tripeptidyl-Peptidase II

The oligopeptidase tripeptidyl-peptidase II (TPP II) is up-regulated Burkitts lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracycline-regulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells.

Chromosomal breaks during mitotic catastrophe trigger γH2AX–ATM–p53-mediated apoptosis

Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the γH2AX–ATM–p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR (ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 14-3-3σ (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.

Down regulation of 3p genes, LTF, SLC38A3 and DRR1, upon growth of human chromosome 3-mouse fibrosarcoma hybrids in severe combined immunodeficiency mice.

We have applied a functional test for tumour antagonizing genes based on human chromosome 3 (chr3)–mouse fibrosarcoma A9 MCHs that were studied in vitro and after growth as tumours in severe combined immunodeficiency (SCID) mice. Previously, we reported that 9 out of the 36 SCID‐tumours maintained the transferred chr3 (“chr3+” tumours), but lost the expression of the known human TSG fragile histidine triad gene (FHIT) in contrast to 14 other 3p‐genes examined. Here we report the results of the duplex RT‐PCR analysis of 9 “chr3+” tumours and 3 parental MCHs. We have examined the expression of 34 human 3p‐genes from known cancer‐related regions of instability, including 13 genes from CER1 defined by us previously at 3p21.33–p21.31 and 10 genes from the LUCA region at 3p21.31. We have found that in addition to FHIT, expression of the LTF gene from CER1 at 3p21.33‐p21.31 was lost in all 9 tumours analyzed. The transcript of the solute carrier family 38 member 3 gene (SLC38A3) gene from LUCA region at 3p21.31 was not found in 8 and was greatly reduced in 1 out of these 9 tumours. Expression of the down‐regulated in renal cell carcinoma gene (DRR1) gene at 3p14.2 was lost in 7 and down regulated in 2 “chr3+” tumours. In the SCID‐tumour derived cell lines treatment with 5‐aza‐2′‐deoxycytidine restored the mRNA expression of LTF, indicating the integrity of DNA sequences. Notably that transcription of the LTF and 2 flanking genes, LRRC2 and TMEM7, as well as transcription of the SLC38A3 gene, were also impaired in all 5 RCC cell lines analyzed. Our data indicate these genes as putative tumour suppressor genes.

Microcell-mediated chromosome transfer provides evidence that polysomy promotes structural instability in tumor cell chromosomes through asynchronous replication and breakage within late-replicating regions

It was reported earlier that normal chromosome 3 (chr3) transfer into tumor cells of different origin may suppress their ability to grow in SCID mice. Tumorigenicity may be restored by the loss of certain 3p regions. We transferred a normal cell‐derived chr3 into cells of a human renal cell carcinoma line and followed the chromosomal changes during in vivo and in vitro growth. In cells cultivated for 6 weeks or more and in the tumors grown in SCID mice, supernumerary chrs3 were always rearranged, accompanied by 3p losses. Unexpectedly, we found that the rearrangements affected not only the transferred exogenous chr3, but also the endogenous chrs3. Other chromosomes that were polysomic in the recipient cells were affected as well, suggesting that polysomy may be associated with structural chromosome instability. The dominant chromosomal aberrations were unbalanced translocations with preferentially pericentromeric breakpoints. The breakpoint distribution on chr3 preferentially affected the pericentromeric 3p11 (8 breaks) and 3p12–13 (5 breaks) regions. The regions 3p14 and 3q26–27 occasionally were involved as well (one break in each case). These four regions were the latest replicating, as shown by BrdU incorporation–based replication banding. Using fluorescence in situ hybridization–based replication timing, we detected asynchronous and incomplete centromere replication in cells with 3 or 4 copies of chr3, but not in cells with 2. We concluded that in tumor cells, asynchronous and incomplete replication of polysomic chromosomal parts is associated with aberrations that have breakpoints within the late‐replicating regions. This may explain the increased structural chromosome instability and preferential pericentromeric localization of breakpoints in hyperploid tumors.

Interstitial deletions including chromosome 3 common eliminated region 1 (C3CER1) prevail in human solid tumors from 10 different tissues

A human chromosomal segment regularly lost during tumor formation of microcell hybrids in SCID mice has been mapped to 3p21.3. This segment, called chromosome 3 common eliminated region 1 (C3CER1, also referred to as CER1), may harbor multiple tumor‐suppressor genes. Because it was found that similar regions were eliminated in an inter‐ and intraspecies system and in two tumor types (mouse fibrosarcoma and human renal cell carcinoma), we hypothesized that the importance of C3CER1 would transgress tissue specificity, that is, it could occur in tumors derived from multiple tissues. To evaluate the loss of C3CER1 in various human tumor types, we conducted loss of heterozygosity (LOH) analysis of 576 human solid tumors from 10 different tissues and compared the frequency of deletion in the C3CER1 area to that in two other regions on 3p: the FHIT/FRA3B region, at 3p14.2, and the VHL region, at 3p25.3. Deletions were detected in the C3CER1 region in 83% of informative tumors. Half (47%) the LOH‐positive tumors showed LOH at all informative markers, indicating a large deletion. The other half (53%) had a discontinuous LOH pattern, suggesting interstitial deletions or breakpoints. The proportion of tumors with C3CER1 deletions was high in all tumor types investigated, ranging from 70% to 94%, except for the soft‐tissue sarcomas (40%). In the VHL and FHIT regions, deletions were observed in 73% and 43%, respectively, of the tumors. Of the three 3p regions analyzed, the highest deletion frequency was observed in the C3CER1 region. Furthermore, we demonstrated that the interstitial deletions including C3CER1 prevail over 3p14.2–pter losses in solid tumors.

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.