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

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

Etude géochimique de quelques calcaires éocènes de la partie nord-ouest du bassin de Transylvanie (Roumanie)

Die chemische, spektrale, roentgenographische und optische Untersuchung einiger Kalksteine aus dem Eozän des Transylvanischen Beckens wird dargelegt. Im untersuchten Gebiet werden zwei geochemische Bereiche unterschieden, die durch die vertikale Verteilung des Strontiums in den Bänken voneinander abweichen.Геохимические исследования некоторых эоценовых известняков северо-западной части Трансильванского бассейна (Румыния) Авторы излагают результаты химического, спекрального , рентгенографического и оптического изучения некоторых эоценовых известняков Трансильванского бассейна. На изученной площади они выделяют два геохимических участка, которые различаются по вертикальному распределению стронция в пластах.The authors present the chemical, spectral, X-ray and optical study of several Eocene limestones of the Transylvanian Basin. Within the perimeter investigated, they distinguish two geochemical compartments differentiated according to the vertical distribution of strontium in the layers.Les auteurs présentent l'étude chimique, spectrale, roentgéno-graphique et optique de quelques calcaires éocènes du Bassin Transylvain. Dans le périmètre étudié, ils distinguent deux compartiments géochimiques qui se différencient selon la répartition verticale du strontium dans les bancs.Imreh Joseph, Mihalka Stephan. Etude géochimique de quelques calcaires éocènes de la partie nord-ouest du bassin de Transylvanie (Roumanie). In: Bulletin du Service de la carte géologique d'Alsace et de Lorraine, tome 23, n°3-4, 1970. Sédimentologie et géochimie de la surface. pp. 163-176

Differences in c-myc and pvt-1 amplification in sewa sarcoma sublines selected for adherent or non-adherent growth

Conversion of solid sarcomas and carcinomas into ascites tumors depends on the in vivo selection of phenotypically altered tumor cell variants that can grow in the dissociated form. Once selected, they retain this property even after prolonged s.c. growth as solid tumors. From an s.c.-passaged subline of an ascites-converted murine sarcoma (SEWA-AS12), we were able to separate cells adapted to the ascites form of growth from cells that can only grow in the solid form on the basis of their differential adherence to palstic. Both c-myc and pvt-1 were amplified approximately 63- to 77-fold in the non-adherent subline (SEWA-AS12-NA), but only 5- to 8-fold in the adherent subline (SEWA-AS12-ADH). This suggests that c-myc and/or pvt-1 amplification may provide a selective advantage to cells that can grow in the dissociated form.Supported by a USPHS grant from the National Cancer Institute, by The Cancer Research Institute, by the Concern Foundation and by a grant from the Gustav V Jubilaeum Fund. J. Minarovits was a recipient of a fellowship from the Swedish Cancer Society

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21,1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.This work was supported by grants from the Swedish Cancer Society, the Cancer Research Institute/Concern Foundation, and Karolinska Institut

Profiling of copy number variations (CNVs) in healthy individuals from three ethnic groups using a human genome 32 K BAC-clone-based array

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was -350 kb and involved in total -117Mb or -3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics