Cytogenetic studies of tumor tissue from patients with nonfamilial renal cell carcinoma.

A method combining an enzymatic technique and short term culture was applied to 27 tumor tissues from 22 patients with nonfamilial renal cell carcinoma in order to establish the chromosome changes in these tumors. Chromosome analyses were successfully carried out in quinacrine mustard-Hoechst 33258 and G-banded preparations of 14 tumors from 12 patients, including 2 cases in which established cell lines were obtained after 43 and 64 days in culture and maintained for 25 and 30 passages in an in vitro system, respectively. The modal chromosome numbers ranged from 38-46 in 11 samples, involving chromosomes in structural and numerical changes and 72 chromosomes in one case, with the remaining 2 samples showing a variety of chromosome numbers. Banding analysis revealed 45 clonal aberrations in 11 tumor samples from 10 patients and nonclonal aberrations in the remaining 3 samples from 2 of the patients. Rearrangements of chromosome 3 were observed in 12 tumors, with the breakpoints on this chromosome almost totally clustered from p11 to p21. In one case both primary and metastatic tumors were studied, and an isochromosome for the long arm of chromosome 1 was observed as clonal in origin in the metastatic tissue. Two cases showed nonclonal changes. The remaining case had one clonal abnormality, i.e., deletion of 6q. Of the remaining 33 clones, chromosomes 1, 2, 6, 11, and 17 were frequently involved. These results suggest that renal cell carcinoma may be cytogenetically classified into 3 categories: (a) tumors with changes of chromosome 3: (b) tumors with other clonal aberrations; and (c) tumors without clonal changes. Rearrangements of chromosome 3 may be possibly associated with the genesis and/or progression of renal cell carcinoma.

Isolation of mouse T-cell lymphoma lines from different long-term interleukin 2-dependent cultures.

A number of different biological properties have been ascribed to the hormone-like protein interleukin 2 (IL-2). However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice. Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays. Sustained log-phase growth of these T-cells in vitro has been achieved using Petri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate. The IL-2-independent line grew as a T-cell lymphoma when injected i.p. into pristane-treated, but not untreated, syngeneic C57BL/6 mice. In contrast, cells from the IL-2 parental line CTLL-2 did not grow in vivo. Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-). Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident. Moreover, karyotypic analysis using a quinacrine:Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines. IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6. Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

Suppression of tumorigenicity in three different cell lines of human oral squamous cell carcinoma by introduction of chromosome 3p via microcell-mediated chromosome transfer.

It has been suggested that loss of the short arm of chromosome 3 is one of the most frequent abnormalities in human head and neck cancers including oral squamous cell carcinomas (SCC) and that one or more putative tumor suppressor gene(s) which may contribute to the initiation and/or progression of these tumors might be located on chromosome 3p. In this study, we examined the effects of introducing human chromosome 3 or 7 by microcell hybridization on the tumor-associated phenotypes of three different human oral SCC cell lines, HSC-2, HSC-3 and HSC-4. Transfer of a single chromosome 3p completely suppressed the tumorigenicity of all three parental cell lines, which showed a significant decrease in growth rate in vitro and morphological changes. In contrast, transfer of chromosome 7 had no effect on HSC-2 and HSC-4 cells, although it suppressed the tumorigenicity of HSC-3 cells without modifying their in vitro growth properties. Our findings provide additional confirmatory evidence that loss or inactivation of putative tumor suppressor gene(s) present on chromosome 3p might be primarily involved in the development of human oral SCC. The possibility that chromosome 7 may carry another tumor suppressor gene(s) is also discussed.

Cryptic insertion and translocation or nondividing leukemic cells disclosed by FISH analysis in infant acute leukemia with discrepant molecular and cytogenetic findings.

Of 51 infants with acute leukemia, 13 (25%) had contradictory findings on 11q23/MLL rearrangements that were analyzed by cytogenetic and Southern blot methods: seven had rearranged MLL and normal karyotype, four had rearranged MLL and abnormal karyotype with no 11q23 translocation, and two had germline MLL and 11q23 translocations. Fluorescent in situ hybridization (FISH) analysis using an MLL probe that was performed to elucidate the discrepancy disclosed the presence of normal dividing cells and nondividing leukemic cells in the same bone marrow in five patients, and cryptic insertion or translocation in another five. Subsequent FISH and reverse transcription-polymerase chain reaction analysis identified the MLL-AF10, MLL-AF4, or MLL-AF1q fusions that were produced by the cryptic rearrangements in four of the five patients. In the remaining three patients, the breakpoint of 11q23 translocation was located distal to the MLL locus in one, and the discrepancy was unresolved in two. Thus, FISH should complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in infant leukemia, and when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.

Molecular Evidence for Convergence and Parallelism in Evolution of Complex Brains of Cephalopod Molluscs: Insights from Visual Systems.

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called "evolutionary tinkering" or "co-option", and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the "camera" eye from nautilus' "pinhole" eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids' opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Influence of strain difference on the karyotypic changes in N-nitroso-N-butylurea--induced mouse lymphomas.

Karyotypes were analyzed in 44 cases of mouse lymphomas induced by continuous oral administration of N-nitroso-N-butylurea (NBU): 14 cases of C57BL/6JKok, 6 from LT/sv, 5 from MT/Hok, 2 from (AKR/MsHok x T1Ct)F1, 2 from (AKR/MsHok x CBA/H-T6Hok)F1, and 15 from the first backcross generation of (CBA/Hok x CBA/H-T6Hok)F1 to CBA/Hok. Thirty-six were thymic lymphomas (TLs) and the remaining 8 nonthymic lymphomas (NTLs). No chromosome abnormality was found in 8 NTLs, whereas modal cells were karyotypically changed in 13 TLs. Unlike the situation in NTLs, chromosomally abnormal cells were always present in the remaining 23 TL cases in which modal cells showed no apparent deviation from the constitutional host karyotype. A total of 31 independent karyotypic changes were identified in 26 cytogenetically abnormal major clones from 19 TLs. Although trisomy 15 was the most frequent karyotypic change, being present in 9 TLs, its incidence was substantially low compared with experimentally induced murine TLs so far reported. Furthermore, 6 of the 9 trisomy 15 clones clustered to (CBA x T6)F1 x CBA. Thus, it seems probable that the genetic background of the host animals has a profound influence upon the frequency of trisomy 15 in TLs.

Recurrent endometrial adenocarcinoma with rearrangement of chromosomes 1 and 11.

A chromosome study was performed in a recurrent endometrial cancer. The cytogenetic analysis performed with a quinacrine-Hoechst banding technique revealed rearrangements of chromosomes #1 and #11 [i.e.: der(11),t(1;11)(q21;q23)], which was found in all metaphases. In addition, a deletion of chromosome 6(q21), trisomy of chromosomes #7 and #10, monosomy X, and -4 as the clonal changes. Partial trisomy for a long arm of chromosome #1 also was observed in almost all analyzed metaphases (11 of 12 cells). These data showed the association of rearrangement of 1q in endometrial cancer and/or redevelopment of tumor, and also suggest the possible participation of chromosome #11 on which the human proto-oncogene c-ets was mapped.

14q+ in Ph-positive chronic myelogenous leukemia.

Two cases of chronic myelogenous leukemia with a Ph translocation and an additional chromosome change of the long arm of a chromosome #14 (14q+) are reported. The breakpoints on chromosome #14 were identified as 14q24 and 14q32, respectively. One of the patients did not show any evidence of blastic transformation; the other patient developed a myeloid blastic crisis when the abnormal 14q+ was seen in the bone marrow cells.

Involvement of chromosomes 4, 11, and 17 in a case of myelodysplastic syndrome.

A case of myelodysplastic syndrome in a 68-year-old male in whose marrow cells two translocations were established, i.e., t(4;11)(q13;q23) and t(11;17)(p?:q11), as well as other karyotypic changes (-6,-18,15p+), is described. The relation and identity of the bands involved in the translocations affecting chromosomes #11 and #17 in leukemias in which these chromosomes are specifically affected, i.e., t(4;11) in acute myelomonocytic leukemia and t(15;17) in acute promyelocytic leukemia, are discussed in relation to the case described.

Functional evidence for involvement of multiple putative tumor suppressor genes on the short arm of chromosome 3 in human oral squamous cell carcinogenesis.

Cytogenetic and restriction fragment length polymorphism (RFLP) analyses have suggested that a putative tumor suppressor genes(s), which may play an important role in the development of human oral squamous cell carcinoma (SCC), is located on the short arm of chromosome 3 (3p). We previously reported that introducing in intact human chromosome 3 into three different oral SCC tumorigenic cell lines completely suppresses the tumorigenicity of each cell line with significant decrease in the in vitro growth rate and morphological changes. To map the tumor suppressor gene(s) on 3p, we have now examined the tumorigenicity of microcell hybrid clones containing various fragments derived from 3p that were introduced by microcell-mediated chromosome transfer. Sixteen hybrid clones were obtained from four successful experiments, and these clones were classified into two groups: 4 fully tumorigenic clones and 12 suppressed phenotype clones. Analyses of the 3p segments in the series of hybrid clones with the use of RFLP or microsatellite markers revealed that the 3p21.2-p21.3 or 3p25 regions or both were consistently retained in the 12 clones with suppressed phenotype but not in the 4 tumorigenic clones. The more proximal 3p13 region also was retained in three nontumorigenic clones. The overall results are fairly compatible with recent evidence that there are three discrete regions on 3p showing frequent allelic losses on oral SCC, and they directly provide functional evidence for the presence of tumor-suppressor genes for oral SCC in these regions. The possibility that three genes, FHIT, VHL, and T beta R-II, recently identified on 3p may be significantly involved in oral SCC development is also discussed.

Translocation (9;22)(q22;q12). A recurrent chromosome abnormality in extraskeletal myxoid chondrosarcoma.

Cytogenetic analysis of a case of extraskeletal myxoid chondrosarcoma revealed a reciprocal translocation between 9q and 22q in almost all metaphases analyzed. Structural rearrangements involving 9q and 22q have been reported previously in three cases of extraskeletal myxoid chondrosarcoma. The breakpoints on chromosomes 9 and 22 in the present case were in regions 9q22-q31 and 22q11-q12.2, respectively. The same breakpoints were present in all three previously reported cases. Thus, this recently identified rearrangement of 9q and 22q may serve as a critical cytogenetic parameter for the diagnosis and classification of extraskeletal myxoid chondrosarcoma, as well as being a primary chromosomal event in the course of development of this rare malignancy.

Chromosome changes in mycosis fungoides in an XYY male.

A 32-year-old white male was diagnosed as having mycosis fungoides in 1976; bone marrow biopsy and aspiration in August 1984 revealed infiltration with neoplastic cells. Cytogenetic analysis of the cells from the bone marrow specimen showed that 48 of 50 metaphases contained an extra Y chromosome (i.e., 47,XYY). The remaining two cells were hypotetraploid and hyperpentaploid, respectively, with a common marker derived from chromosome #2. The metaphases obtained by PHA stimulation of peripheral blood cells showed a 47,XYY pattern. An interleukin 2 (IL-2) dependent T-cell line was established from the patient's blood mononuclear cells; all metaphases of this line had an extra Y chromosome. Thus, this case is one of a mycosis fungoides developing in an XYY male.

Two 14q+ chromosomes in malignant lymphoma: crucial cytogenetic changes on 14q.

We encountered a 36-year-old white male patient with poorly differentiated lymphocytic lymphoma, whose lymph node cells showed a clonal cytogenetic change involving chromosome #14, i.e., 47,XY, + 2,der(14),t(14;14)(14pter----14q32;14q24----14q32++ +). In addition to this change, cells with a translocation between chromosomes #2 and another #14 [t(2;14)(q21;q24)], as well as a missing chromosome #8 were found. We have reviewed the literature dealing with two or more changes affecting chromosome #14 and discussed the importance of the cytogenetic change at band 14q32 in malignant lymphoma.

Cytogenetic changes at 11q11, 11q23, and 17q11 in myelodysplastic syndrome.

Cytogenetic studies were performed on two patients with myelodysplastic syndromes. One patient was a 68 year old Japanese male in whose bone marrow cells two translocations were established, i.e., t(4;11)(q13;q23) and t(11;17)(q11?;q11), as well as other karyotypic changes (-6,-18,15p+). The other patient was a 74 year old white male whose bone marrow cells showed six marker chromosomes, i.e., der(5),t(5;17)(q12;q11), der(6),t(6;5)(q27;q22), der(8),t(8;11;?)(q11;q11----q23;?), der(11),t(11;?)(q11;?), an isochromosome of the long arm of chromosome #8, and a small G-group sized marker chromosome of unknown origin. Though the translocation patterns in the abnormal cells in these two cases were different, the breakpoints of the marker chromosomes were almost the same, i.e., 11q11, 11q23, and 17q11. Also, changes of chromosome #6 were observed; the first case showed monosomy 6 and the second a 6q+ marker chromosome. In these two cases of myelodysplastic syndromes, common sites of chromosome breakage and reunion of 11q23 and 17q11 were close to recently established sites of human cellular oncogene homologs, c-ets (11q23) and c-erbA (17q21----24). These associations draw attention to a possible relationship between chromosome changes in myelodysplastic syndromes and oncogene (or other gene) activation and/or dysfunction.

A case of chromophobe renal cell carcinoma associated with low chromosome number and microsatellite instability.

Cytogenetic study in a case of a human chromophobe renal cell carcinoma revealed a hypodiploid chromosome number of 36 with loss of chromosomes 1, 2, 5, 6, 10, 13, 15, 17, 21, and X. The tumor DNA showed microsatellite instability in dinucleotide repeat microsatellite markers. This is the fourth case that has been fully karyotyped and showed a low chromosome number in a chromophobe renal cell carcinoma. Our data in the present study are consistent with those in the literature. It is suggested that human chromophobe renal cell carcinoma may possibly be characterized by tumor cells with low chromosome number or microsatellite instability.

Four cases with complex Philadelphia translocations, including one with appearance de novo of a "masked" Ph.

Four cases of chronic myelogenous leukemia (CML) with complex Philadelphia (Ph) translocations are described. The first case was that of a 50-year-old woman in the chronic phase of CML. Her leukemic cells showed a complex Ph translocation involving chromosomes #9, #11, and #22 [i.e., t(9;9;22;11)(11qter----11q11::9q11----9q34:: 9p11----9pter;22qter----22q11::9q34?;11 pter----11q11::22q11----22qter)]. In addition to the complex Ph translocation, the leukemic cells contained del(10)(p13). The second case was that of a 21-year-old man whose leukemic cells contained a translocation involving chromosomes #5, #9, and #22 [i.e., t(5;22;9)(q31;q11;q34)], resulting in a "masked" Ph chromosome. The third case was that of a 37-year-old man whose leukemic cells had a complex Ph translocation involving chromosomes #8, #9, and #22 [i.e., t(8;9;22)(q13;q34;q11)]. The fourth patient was a 41-year-old woman diagnosed as having CML in myeloid blastic phase, at which time the first specimen was examined by us. This blood sample showed a karyotype of 45,XX, -9, -17, -22, +mar1, +mar2,9q+. No Ph chromosome was present. A standard Ph translocation was detected in the cells obtained from the spleen, when the patient underwent splenectomy for treatment of the blastic crisis. Subsequent specimens obtained from the blood and bone marrow showed that the leukemic cells contained three clones: 45,XX, -9, -17, -22, +mar1, +mar2,9q+/46,XX, -17, +mar1,t(9;22)(q34;q11)/46,XX,t(9;22)(q34;q11). Cells with the "masked" Ph chromosome were thought to have been derived from the clone with the standard Ph translocation. We postulate that some variant Ph translocations, including those with a "masked" Ph chromosome, may be generated by a stepwise process following the genesis of a standard Ph translocation.

Rearrangement of chromosome 3 in renal cell carcinoma.

Rearrangements involving chromosome #3 were detected in 8 of 12 nonfamilial renal cell carcinomas. These results suggest that rearrangement of chromosome #3 is associated with the genesis and progression of a subclass of human renal cell carcinoma.

Chromosome rearrangements at 12q13 in two cases of chondrosarcomas.

We analyzed the karyotypes of two moderately differentiated (grade 2) chondrosarcomas. Case 1 had a reciprocal translocation between chromosomes 6 and 12, t(6;12)(q25;q13) in most of the cells analyzed, as well as trisomies of chromosomes 7, 8, 11, 17, 19, and 21 and tetrasomy of chromosome 19. A reciprocal translocation involving chromosomes 12 and 19, t(12;19)(q13;q13), was noted as a highly clonal abnormality in the other case. Some cells had t(12;19) as the sole chromosome abnormality. Thus, chromosome rearrangements involving the long arm of chromosome 12 at the same region (q13) were commonly identified in the two tumors. These findings suggest that the rearrangements at 12q13 are nonrandom acquired changes that characterize a subgroup of chondrosarcomas.

Homozygous deletions on the short arm of chromosome 3 in human oral squamous cell carcinomas.

Recent cytogenetic and allelic deletion analyses have demonstrated that deletions on the short arm of chromosome 3 (3p) are frequently found in various cancers, including oral squamous cell carcinomas (OSCCs). This suggests that one or more tumor suppressor gene(s) for these malignancies might be located on 3p. In the present study, to further define the region(s) on 3p that harbor putative tumor suppressor gene(s) for OSCCs, we have investigated the existence of homozygous deletions (HDs) at 34 loci on 3p, in 14 OSCC cell lines. HDs were detected within the FRA3B region at 3p14.2 in only two cell lines (HSC-4 and TSU). Recently, the human fragile histidine triad (FHIT) gene was isolated from this region, abnormalities of which have been found at high frequencies in several types of human cancers. We also examined the expression of the FHIT gene, using reverse transcription-polymerase chain reaction (RT-PCR) and exon-specific PCR, in the two OSCC cell lines which showed HDs at 3p14.2. There was no detectable expression of exon 5, which was the first protein-coding exon of FHIT gene, in HSC-4 cells, indicating that this region was homozygously deleted in this cell line. On the other hand, HD in the TSU cells did not affect the coding region of the FHIT gene, and the wild-type transcript was detected by RT-PCR. Therefore, several candidate tumor suppressor genes, including the FHIT gene, may reside in these homozygously deleted regions. To our knowledge, this is the first report of HDs on 3p in OSCCs.

Inactivation patterns of the p16 (INK4a) gene in oral squamous cell carcinoma cell lines.

To determine whether inactivation of the p16 gene mapped to the chromosome 9p21 region is associated with the development of oral squamous cell carcinoma (SCC), we investigated the mutational states of two forms of alternative transcripts (alpha and beta) from the p16 gene in 14 oral SCC cell lines by means of RT-PCR, PCR, direct sequencing and methylation analyses. Alterations of the alpha transcript were detected in all of the cell lines examined: homozygous deletions in three lines; subtle mutations in exons 1 alpha or 2 in four lines; skipping of exon 2 in two lines; hypermethylation of the 5' CpG island of the p16 gene in four lines; and an unknown mechanism in one line. On the other hand, abnormalities of the beta transcript were observed in seven of the 14 cell lines. Nonetheless, the mutations that essentially affect the function of the encoded protein were found only in five cell lines, including three lines with homozygous deletion. There was no cell line having only beta transcript alterations. Thus, alteration of the alpha transcript of the p16 gene was a highly frequent event in oral SCC. Since this type of alteration resulted in gene inactivation through multiple pathways, it may play a major role in the process of oral SCC development.