A microcell hybrid based "elimination test" in the search for malignancy related genes

Sammanfattning: A microcell hybrid based "elimination test" in the search for malignancy related genes Stephan Imreh This thesis is focused on the elimination test developed by us (Imreh et al, 1994). The elimination test was designed as a functional test for malignancy related gene identification. Its task is to follow the cytogenetic and molecular alterations on single normal chromosomes transferred into tumor cells. We assume that these modifications when occur regularly may be connected to selective growth advantage providing functions. Paper 1. In our first series of experiments we used MCH903.1, MCH906.8 carriing a cytogenetically intact human chr 3 and MCH910.7, MCH939.2 and MCH924.4 carriing human chromosome 3 that contained the deletions, del(3)(pter p21.3), del(3)(p21.3-p14) and 3del(p24-p14)(q21-q26). We initiated the first quantitative/functional study by FISH chromosome painting on tumors observing that two normal human chr.3 / mouse MCHs lost a cytogenetically intact form of the introduced chr.3 during progressive growth in SCID mice. Only fragments translocated into mouse chromosomes (chimeric translocations) were maintained. PCR-analysis using 20 pairs of primers had revealed a common eliminated region including four markers spanning approx. 40 cM on the 3p24-p21.3 region: THRB (3p24.1 p22), AP20R (3p21.33), D3S32 (3p21.3-p21.2) and D3S1029 (3p21.31-p21.2) in 20 SClD-mouse tumours derived from chr3 / mouse MCHs. Control microcell hybrids with chr 1 and 8 were not fragmented during SCID tumor growth. Based on this results we proposed the "elimination test" as a supplementary method for tumor suppressor gene identification. We expected that it may provide a simpler and more easily interpreted test than direct suppression of tumorigenicity that may be hampered by the accidental loss or mutation of an introduced gene. Paper ll. In order to further narrow down the common eliminated region and to characterize the losses, five MCH lines that carried human chr. 3 were included in a follow up study. MCH903.1, MCH906.8 and MCH910.6 carried a cytogenetically intact human chr 3, MCH910.7 and MCH939.2 carried human chr 3 that contained the deletions, del(3)(pter-p21.3) or del(3)(p21.3-p14), respectively. MCH901 carried a cytogenetically intact human chr 1, MCH 240.3 carried 1-3 copies of a cytogenetically intact human chr 13, MCH203.4 carried two copies of human chr 13 as Robertsonian fusion chr (translocated into a mouse chromosome), MCH313.4 carried a cytogenetically intact human chr 17 and MCH904.11 carried a cytogenetically intact human chr 8. MCH-derived SClD-mouse tumors, 22 new and 5 previously reported were analysed by fluorescence in situ hybridisation (FISH: direct painting, DP and reverse chromosome painting, RP), Southern blotting and PCR. 53 chr. 3-specific markers were tested for the presence by PCR. The common eliminated region designated as CER has been narrowed down from an estimated average spacing of 40 cM to ~7 cM. CER included the markers AP20R (3p21.3), D3S966 (3p21.3-p21.3), D3S1029 (3p21.3-p21.2) Wl-7947 (3p22 p21.3), D3S2354 (3p22-p21.3), D3S32 (3p21.3-p21.2, and B362WB9 (3p21.3-p21.2), and was bordered distally by the D3S1260 marker and proximally by the D3S643 marker. The delineation of 7 cM CER was possible due to the identification (both by PCR and RP of an interstitially retained 3p21.3-21.2 fragment designated later as "rebox-l"and containing 7 markers on the centromeric verge of the CER. A significant solid tumor LOH cluster concorded with the CER.. The known HDs in SCLC lines seemed to flank telomerically and centromerically the CER. Paper lll. In 24 serially passaged SCID derived tumors of MCH910.6 and 906.8 (originally containing intact chr.3) the previous results on the elimination of 3p21.3 segments were confirmed. The "rebox 1" was maintained in tumors but on the telomeric side of the CER, another retained box was found: the "rebox-A". In 10 MCH 910.6 derived tumors one or two double minute (dmin) like fragments were observed. By PCR analysis using 24 markers we covered the the interval between D3S1611 and D3S1235 (3p22-21.2). D3S32 and D3S2354 are regularly eliminated during in vivo tumor growth whereas the other 22 markers D3S1611, ACM, D3S1260, Wl-692, D3S2343, D3S966, D3S1029, D3S643 Wl-2420, MST1, GNAI2, D3S1235, D3S1298, GLB1, Wl-4193, D3S3658, D3S3559, D3S3678, Wl-6400, Wl-7947, B362WB9 and D3S10865 are regularly retained. We have defined a common eliminated region (designated as CER1) of approximately 1.6 cM, inside the previously identified CER. CER1 extends to D3S1029 on the telomeric and D3S643 on the centromeric side. Paper IV While pursuing these studies, we have noticed that segments on the long arm of chr. 3 are frequently retained after SCID mouse passage. Using DP, RP and PCR and 4 successive SCID mouse passages of chr.3 MCHs concordant results were obtained in the majority of tumors. MCH903.1: intact chr. 3, MCH910.8: del(3)(pter-p21.3); MCH939.2: del (3)(p22-p14); MCH939.3:del(3)(pter-cen). The chr. 3 donors were two normal human diploid fibroblast lines, HFDC (for MCH903.1 and 910.7 designated as A1, A2) and HHW1108 (for MCH939.2 and 939.3 designated as B1, B2). We have confirmed the preferential loss of 3p and showed that after four SCID mouse passages 8 markers (D3S1282, GLUT2, D3S1262, D3S1314, SST, 924ZO69R, 924ZO66R and D3S1265) are obstinately retained in 17 tumors and 9 single cell clones. There is a common retained region (CRR) on 3q25-qter. Several oncogenes including FIM3, EVI1, BCL6, ETS1, ERM are located within CRR. A remarcable concordance was found between the location of CRR and segmental gains over the same region observed by CGH in uterine cenvix carcinoma, ovarian cancer and small cell lung carcinoma. Paper V Early in our studies on the role of chr.3 chromosome aberrations in cancer initiation, by radioactive in situ hybridization we have localised the D3F15S2 marker to 3p21.2.p21.1, telomeric to the familial RCC t(3;8) translocation breakpoint concluding that the region affected by this translocation is not identical with the region of 3p most frequently deleted in sporadic RCC.