Stephan Imreh

Search for unknown tumor-antagonizing genes.

Following the ingenious prediction of Alfred Knudson in 1971, the first tumor suppressor gene, RB1, has been isolated. Its product, the RB1 protein, was found to play a major role in the control of the cell cycle. The loss of heterozygosity (LOH) technique, introduced by Cavenee and colleagues, was an important milestone toward the confirmation of Knudsons hypothesis and the identification of the gene. Subsequently, the LOH technique has provided important clues that have led to the discovery of other tumor suppressor genes. Most of them play important roles in the regulation of the cell cycle and/or of apoptosis. Circumstantial evidence suggests that still other and perhaps many unknown genes may participate in the protection of the organism against malignant growth. The numerous genome losses in tumors, detected by LOH, comparative genomic hybridization, and by cytogenetic techniques, support this possibility. The early work of one of us (G.K.), together with Henry Harris and Francis Wiener, had shown that the malignant phenotype can be suppressed by hybridizing malignant with low‐ or non‐tumorigenic cells. However, analysis of this phenomenon failed to assign the inhibition of tumorigenicity to any particular gene. We have pursued the search for new tumor‐antagonizing genes with two unconventional approaches, focusing on human chromosomal subband 3p21.3, a region frequently targeted by cytogenetically detectable deletions. We have detected four clusters of candidate tumor suppressor genes at 3p21.3 by a combination of deletion mapping and the “elimination test.” These findings raise the question whether the number and variety of genes that may contribute to the defense against uncontrolled proliferation may have been underestimated.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its β-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK−)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first- generation tumours when L-M(TK−) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

A 3p21.3 region is preferentially eliminated from human chromosome 3/mouse microcell hybrids during tumor growth in SCID mice

We have previously shown that four markers spanning the 3p24‐p21.3 region, THRB, AP20R, D3S1029, and D3S32, were regularly eliminated from three human chromosome 3 (chr3)/mouse microcell hybrids (MCHs) during tumor growth in SCID mice. In an attempt to narrow down the eliminated region, we have studied 22 new SCID mouse tumors derived from 5 MCH lines carrying human chr3. They were analyzed by fluorescence in situ hybridization (FISH), Southern blotting, and polymerase chain reaction (PCR). MCHs that carried human chr1, chr8, chr13, and chr17 were examined as controls. We could identify a common eliminated region (CER) at 3p21.3, bordered distally by D3S1260 and proximally by D3S643/D3F15S2. Eight of 53 chr3‐specific PCR markers, AP20R, D3S966, D3S3559, D3S1029, Wl‐7947, D3S2354, AFMb362wb9, and D3S32, were localized within the CER. This finding is consistent with the notion that a tumor suppressor gene may be located in this area, as suggested by frequent loss of heterozygosity (LOH) within this region observed in several types of solid tumors. Genes Chromosom. Cancer 18:200–211, 1997.

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.

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).

Mapping of a new MAP kinase activated protein kinase gene (3PK) to human chromosome band 3p21.2 and ordering of 3PK and two cosmid markers in the 3p22–p21 tumour-suppressor region by two-colour fluorescencein situ hybridization

ANotl-linking clone NL1-210 (D3S1656) that contains the human MAP kinase activated protein kinase (3PK) gene was localized to 3p21.2 on DAPI-banded and propidium iodide (R-bands)-stained chromosomes by fluorescencein situ hybridization (FISH). For more precise localization of 3PK, two cosmid probes were used as a frame. In order to establish this frame, twoNotl-linking clones, NL2-008 (D3S1648) and NL3-003 (D3S3872) were used to screen the cosmid library for locus extension. They mapped to 3p21 and were found to belong to two separate contigs ofNotl-jumping and linking clones. Using FISH on DAPI-banded metaphase chromosomes, we have determined the precise localization of cosNL2-008 and cosNL3-003 to 3p21.2–p21.1 and 3p22–p21.3 respectively. The 3PK gene was localized to the 3p21.2 region within this frame by two-colour FISH. The orientation of the probes are tel-D3S3872-3PK-D3S1648-cen.

Spontaneous development of plasmacytomas in a selected subline of BALB/cJ mice

Sixty per cent of BALB/cAnPt mice injected intraperitoneally (i.p.) with tetramethylpentadecane (pristane) develop plasmacytomas (PCs), whereas less than 10% of BALB/cJ develop such tumours. Most other mouse strains are completely resistant. Resistance is dominant over susceptibility in F1 hybrids between BALB/cAnPt and the resistant non-BALB/c strains, suggesting that susceptibility may be due to some genetic defect. (BALB/cAnPtxBALB/cJ)F1 hybrids have a PC incidence of 36-42%. Previously, BALB/cJ has been shown to harbour at least one resistance gene (Potter et al., Genomics 1988, Vol. 2, pp. 257-262). On the assumption that BALB/cJ may contain a segregating resistance gene, we cross BALB/cJ females with pristane-pretreated BALB/cJ males that were found to be carrying PC cells intraperitoneally 5-7 months after pristane treatment. After two selective crosses, 62% of the BALB/cJ subline BALB/cM2/22 developed PC after pristane and 52% after pristane followed by Abelson virus, while unselected controls had an incidence of 11% and 0%, respectively. Moreover, six spontaneous plasmacytomas developed in untreated females of the selected colony. Five of these carried T(12; 15) (F2; D2/3) translocations. The sixth had a T(1; 10) (G; C1) translocation and an interstitial duplication of segment (C1/E3) on one chromosome 5. It may be concluded that pristane treatment is not a prerequisite for the induction of the PC associated Ig/myc translocations.

Hypersomy of chromosome 15 with retrovirally rearranged c-myc, loss of germline c-myc and IgK/c-myc juxtaposition in a macrophage-monocytic tumour line

From a lymphoid tumour induced by 7,12-dimethylbenz-[a]-anthracene (DMBA) + methyl-N-nitrose-N-urea (MNU) in an [AKR Rb(6.15) x CBAT6T6]F1 mouse, a macrophage- monocyte line (KT-10) was isolated. Following ethyl methanesulfonate (EMS) treatment, a bromodeoxyuridine (BUdR) resistant subline was selected. Serial propagation of this line in vitro in the presence of BUdR (28 months) with periodic cytogenetic and molecular examinations, has led to the definition of four successive stages. During stage I, the cells were trisomic for chromosome 15. They contained Rb(6.15) and Rb(del6.15) of AKR and T(14;15) of CBA origin. Southern blotting showed the presence of both germline (G) and rearranged (R) c-myc. At stage II, Rb(del6.15) has duplicated. Both Rb(6.15) and T(14;15) persisted together with G-myc and R-myc. In stage III, the CBA-derived T(14;15) was lost, in parallel with G-myc. At this stage, a Dic.In(6.15) was detected. One of its arms was cytogenetically identical with the long arm of In(6.15) in the variant IgK/myc translocations. This chromosome carried R-myc and IgK in juxtaposition, as indicated by comigration on pulsed field electrophoresis (PFGE). At stage IV, the R-myc carrying AKR-derived chromosome 15s were present in six copies. Possible relationships between the increasing R/G myc ratio and changed growth characteristics in vivo and in vitro are discussed.