Biomedical subjects

M Varella-Garcia

Publications and source records attributed to M Varella-Garcia.

At least 37 records · Page 2Linked to original sources

PubMed2000

A novel syndrome of radiation-associated acute myeloid leukemia involving AML1 gene translocations.

AML1 is a transcriptional activator that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. We recently identified 3 patients in whom pancytopenia developed almost 50 years after high-level radiation exposure from nuclear explosions during or after World War II. In all 3 patients, acute myeloid leukemia (AML) eventually developed that had similar characteristics and clinical courses. Cytogenetics from the 3 patients revealed a t(1;21)(p36;q22), a t(18;21)(q21;q22), and a t(19;21)(q13.4;q22). By fluorescent in situ hybridization (FISH), all 3 translocations disrupted the AML1 gene. Two of these AML1 translocations, the t(18;21) and the t(19;21), have not been reported previously. It is possible that the AML1 gene is a target for radiation-induced AML. (Blood. 2000;95:4011-4013)

Acute Disease↗

PubMed2000

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766)

Adenosine Triphosphate↗

PubMed2000

Molecular definition of a small amplification domain within 3q26 in tumors of cervix, ovary, and lung.

A common amplification target encompassing chromosome region 3q25 to q27 has been identified by comparative genomic hybridization analyses in tumors of the cervix, ovary, endometrium, lung, and head and neck. Because this segment spans at least 30 megabases, we undertook a molecular analysis of copy number to more precisely define the amplification domain. Our Southern blot and fluorescence in situ hybridization results with the use of 17 markers confirmed the presence of low-level 3q amplification events in cervical, ovarian, and variant SCLC tumors. Most of the tumor types studied appeared to have similar, broad amplification domains centered within 3q26.2, suggesting that the same target is being affected in all. The ovarian carcinoma cell line NIH:OVCAR3 had a highly restricted amplification domain spanned by four overlapping YAC clones, suggesting a small target. The region of highest amplification included the gene for the RNA component of telomerase (hTR), supporting it as a potential target. Although the importance of low-level amplification is unknown, the consistent and reproducible nature of this event in a variety of carcinomas suggests that 3q26.2 harbors an oncogene whose low-level amplification has a significant influence on tumor biology.

Blotting, Southern↗

PubMed2000

The benzene metabolite, hydroquinone, selectively induces 5q31- and -7 in human CD34+CD19- bone marrow cells.

OBJECTIVE: Chronic exposure to high concentrations of benzene is associated with an increased incidence of myelodysplastic syndrome and acute myelogenous leukemia. Acute myelogenous leukemia developing in patients treated with alkylating agents for other cancers or occupationally exposed to benzene exhibit a pattern of cytogenetic aberrations predominantly involving loss of all or part of chromosomes 5 and/or 7. In contrast, trisomy 8 is observed equally in both de novo and secondary acute myelogenous leukemia. Studies using peripheral lymphocytes or lymphoblastoid cell lines have observed dose-dependent loss of chromosomes 5, 7, and 8 following treatment with the benzene metabolite, hydroquinone. The purpose of this study was to determine the dose response and specificity of hydroquinone-induced aberrations on chromosomes 5, 7, and 8 using human CD34+CD19 bone marrow cells. MATERIALS AND METHODS: Fluorescence in situ hybridization analysis was performed on CD34+CD19- bone marrow cells using the locus-specific probes, 5q31, 5p15.2, and centromeric probes specific for human chromosomes 7 and 8 following hydroquinone exposure. RESULTS: Hydroquinone exposure results in -7, selective deletion of 5q31 but not chromosome 5 and no loss or gain of chromosome 8 in human CD34+CD19- cells. CONCLUSION: CD34+ bone marrow cells are more susceptible and show a different pattern of cytogenetic aberrations as a result of hydroquinone exposure compared to lymphocytes. CD34+ bone marrow cells exhibit unique susceptibility to the development of specific chromosome aberrations that have been identified as the earliest structural changes occurring in the development of secondary myelodysplastic syndrome and acute myelogenous leukemia.

Benzene↗

PubMed2000

Intrachromosomal distribution of telomeric repeats in Eumops glaucinus and Euntops perotis (Molossidae, Chiroptera).

The location of chromosomal telomeric repeats (TTAGGG)n was investigated in two species of the Molossidae family, Eumops glaucinus and Eumops perotis. The diploid chromosome number (2n) is 40 in E. glaucinus and 48 in E. perotis and the fundamental numbers (FN) are 64 and 58, respectively. It has been suggested that the E. glaucinus karyotype has evolved from the E. perotis karyotype through Robertsonian fusion events. In the present study, the telomeric sequences were detected at the termini of chromosomes in both species. In addition, E. glaucinus also displayed telomeric repeats in centromeric and pericentromeric regions in almost all biarmed chromosomes. Conversely, in E. perotis pericentromeric signals were only observed in two biarmed chromosomes. In both E. glaucinus and E. perotis, such telomeric sequences were observed as part of the heterochromatin. The interstitial sites of telomeric sequences suggest that they are remnants of telomeres of ancestral chromosomes that participated in the fusion event.

Animals↗

PubMed1999

Identification of individuals at high risk for head and neck carcinogenesis using chromosome aneuploidy detected by fluorescence in situ hybridization.

To visualize the accumulation of chromosome abnormalities in head and neck squamous cell carcinomas (HNSCC) and investigate the extension of the abnormal field, we applied the fluorescence in situ hybridization (FISH) technique to tumor cells and cells collected from a large extension of clinically normal buccal mucosa distant from the tumor in 10 patients. DNA probes specific for 14 human chromosomes (1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 15, 17, X, and Y) were used in dual-target, dual-color FISH assays. Control specimens were collected from oral mucosa of 10 healthy non-smokers, in order to define the tolerance limits for abnormalities, and from 10 healthy smokers. Extensive aneuploidy was detected in most of tumor specimens, more frequently represented by chromosome gains than losses. Interestingly, the clinically normal distant oral regions displayed chromosomal aneuploidies in seven out of the 10 patients tested. These findings support the occurrence of field cancerization in HNSCC. In addition, interphase FISH is demonstrated as an effective technique for detecting chromosome aneuploidy associated with malignancy and a potential tool for non-invasive screening of individuals at high-risk for HNSCC.

Adult↗

PubMed1999

A dup(17)(p11.2p11.2) detected by fluorescence in situ hybridization in a boy with Alport syndrome.

We describe a de novo dup 17p11 in a boy with Alport syndrome, mild mental retardation, and minor anomalies. Combining classical and molecular cytogenetics analyses, the karyotype was defined as 46,XY.ish dup (17)(p11.2p11.2)(D17S29++,D17S379+). Alport syndrome is associated with mutations in the type IV alpha chain collagen gene, however, no known collagen-related gene is currently mapped to 17p11. Duplications involving 17p11.2 have been reported in Charcot-Marie-Tooth disease, Dejerine-Sottas syndrome, and in a few sporadic patients with mental retardation and minor anomalies, however, no significant clinical similarity was found among these cases and the propositus. Further studies may clarify the meaning of the association between Alport syndrome and duplications of DNA sequences mapped at 17p11.2.

Chromosome Aberrations↗

PubMed1999

Aml1/ETO and Pml/RARA rearrangements in a case of AML-M2 acute myeloblastic leukemia with t(15;17).

We report a case of acute myeloid leukemia FAB-type 2 with a translocation t(15;17)(q22;q12) On the basis of the cytological findings, a translocation t(8;21)(q22;q22) was suspected. FISH analyses using specific probes for t(15;17) and t(8;21) detected both PML/RARalpha and AML1/ETO rearrangements in a few percentage of cells. This case demonstrates the complexities that may occur between cytology and cytogenetic findings and the usefulness of FISH methods to detect an AML1/ETO rearrangement only suspected by cytological examination of bone marrow smears.

Adult↗

PubMed1999

The benzene metabolites hydroquinone and catechol act in synergy to induce dose-dependent hypoploidy and -5q31 in a human cell line.

Chronic exposure to high concentrations of benzene is associated with an increased incidence of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8 and we have previously reported that hydroquinone (HQ) induces deletions of 5, 7 and 8. Benzene metabolism is a requirement for bone marrow toxicity and the phenolic metabolites, HQ and catechol (CAT), have been implicated in benzene hematotoxicity. A research project was designed to determine whether CAT by itself and in conjunction with HQ could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7, and 8 probes we demonstrate that 5 to 150 uM CAT does not produce chromosomal aberrations, however CAT and 25 uM HQ can act in synergy to induce dose dependent loss of these chromosomes. In addition HQ/CAT selectively induces -5q which is not observed for HQ only. These results demonstrate for the first time that CAT/HQ act in synergy to induce specific chromosome loss found in secondary MDS/AML.

Benzene↗

PubMed1998

Chromosomal duplication accompanies allelic loss in non-small cell lung carcinoma.

Hemizygous deletion in the short (p) arm of chromosome 3 is a common finding in non-small cell lung carcinoma (NSCLC) and is postulated to be a crucial early change in lung tumorigenesis. Yet one of the most frequent nuclear abnormalities in both NSCLC and premalignant bronchial epithelium is increase in chromosomal copy number. Deletion and duplication have not been assessed in the same tumor set by both molecular and cytogenetic methods to determine whether allelic loss correlates with chromosomal duplication in the same tumor cell populations. It is also not established what biological mechanisms might lead to allelic deletion and chromosomal duplication. We have investigated changes in the copy number of chromosome 3 in touch preparations of 38 NSCLCs (19 adenocarcinomas and 19 squamous cell carcinomas) using dual-target, dual-color fluorescence in situ hybridization (FISH) assays. Chromosome 3 centromere probe was matched with a 3p14.2 probe [intron 4 of the fragile histidine triad (FHIT) gene] and a 3p21.31 probe (HSemaIV gene). We then correlated FISH results with results of molecular analyses for allelic losses at loci in the regions to which the FISH probes mapped in 20 of these cases. Although various combinations of FISH abnormalities were sometimes detected within the same specimens, individual cases could be classified according to the predominant FISH pattern, usually with one abnormality present in >60% of tumor cells. Chromosomal duplication, indicated by the presence of more than two centromeric signals, was the most frequent abnormality observed by FISH and was accompanied by loss of specific sequences on 3p in approximately one-half of the specimens in which it was observed. The most frequent abnormality observed by molecular analysis was loss of heterozygosity (LOH) in both of the chromosomal regions tested and was demonstrated in 83% of cases with chromosomal duplication. We conclude that LOH may occur in the presence of chromosomal duplication, suggesting that the duplicated chromosome is homozygous. Our findings imply that LOH occurs before chromosomal duplication during lung carcinogenesis.

Acid Anhydride Hydrolases↗

PubMed1998

Malignancy detection by molecular cytogenetics in clinically normal mucosa adjacent to head and neck tumors.

OBJECTIVE: To identify the potential use of chromosome imbalances as biomarkers for tumorigenesis in head and neck squamous cell carcinoma (HNSCC) by fluorescence in situ hybridization (FISH). DESIGN: In this case-control study, chromosome copy numbers were assessed in dual-target, dual-color FISH assays using DNA probes specific for 14 human chromosomes (1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 15, 17, X, and Y) applied to exfoliated epithelial cells. SETTING: University medical center. PATIENTS: We examined 20 cell brushings (from 10 primary tumors and 10 clinically normal margins) collected from 10 patients with HNSCC and compared these with cell brushings from the oral cavity of 10 nonsmoker and 10 smoker control subjects. INTERVENTION: None. MAIN OUTCOME MEASURE: Chromosomal aneuploidy. RESULTS: Specimens from nonsmokers displayed greater than 91% of cells with normal signals, indicating high analytical sensitivity for the probes. Specimens from smokers demonstrated large variability without significant imbalance (P>.05) compared with those from nonsmokers. Tumor specimens from patients with HNSCC displayed significant chromosomal imbalance (P<.05) for all probes except chromosome Y. Similar imbalance, although in lower frequency, was found in all clinically normal adjacent mucosa specimens. CONCLUSIONS: Interphase FISH demonstrated great applicability in detecting chromosome imbalance associated with malignancy in HNSCC and clinically normal adjacent cells, thereby detecting subclinical tumorigenesis. A panel of chromosome probes (chromosomes 3, 8, 9, and 10) is proposed as an efficient and sensitive additional tool for future routine screening of tumor margins and potential diagnosis of residual disease in HNSCC.

Adult↗

PubMed1998

A panel of partial chromosome paints and YAC probes specific for human chromosome 2.

Twenty nine hybrids retaining fragments of human chromosome 2 were characterized by reverse-FISH and by a panel of 106 STSs. Most of the hybrids are radiation hybrids retaining fragments of chromosome 2 as the only human contribution. The hybrid panel dissected chromosome 2 in 69 distinct physical regions, allowing a fine mapping of the sequences. These hybrids are particularly useful as starting points for generation, via Alu-PCR, of specific partial chromosome paints (PCP). We also report the mapping by FISH of 60 YACs located on chromosome 2. These resources can be advantageously used in cytogenetic investigations, with particular reference to cancer cytogenetics, as illustrated with the renal carcinoma cell line KRC/Y.

Chromosome Mapping↗

PubMed1998

Receptor expression, cytogenetic, and molecular analysis of six continuous human glioma cell lines.

Six human glioma cell lines were established from tissues obtained from five patients diagnosed with Kernohan grade IV glioblastoma multiforme and one from a patient with a grade II astrocytoma. One line was from a recurrent patient who had received prior therapy; the other lines were derived from patients at initial diagnosis and/or before cytoreductive therapies other than surgery were given. Considerable variability in phenotypic, karyotypic, and cell surface marker expression was displayed between the six human glioma cell lines. The karyotypes ranged from apparently normal (grade II astrocytoma) to those with complex rearrangements. Trisomy of chromosome 7 was the most common abnormality. The extensive cytogenetic and molecular characterization of these lines may facilitate their utilization in cellular and molecular biologic studies.

Adult↗

PubMed1998

Establishment and characterization of a human glioblastoma multiforme cell line.

Cell lines provide a useful system for further understanding the biology of glioblastoma multiforme. In this study, a new glioblastoma multiforme cell line, GATAGM-96 (Gulhane Askeri Tip Akademisi-Gliblastoma Multiforme-96), was established from a tumor specimen removed from an 80-year-old male patient who underwent surgery for intracranial tumor. Morphologic examination, immunocytochemical staining, growth kinetics, and karyotypic characteristics of this cell line were studied. The cytoskeleton was positive for neuron-specific enolase, vimentin, and neurofilament, and it was negative for glial fibrillary acidic protein, S-100 protein, p53 protein, epidermal growth factor, and transforming growth factor alpha. Growth kinetic studies demonstrated an approximate population doubling time of 38 to 42 h and a colony forming efficiency of 83.3%. The karyotype of the cells demonstrated it as hyperdiploid, with a large subpopulation of polyploid cells. There were numerous structural and numerical chromosome aberrations; most of them were present as clonal events. The phenotypic and chromosomal features detailed on the GATAGM-96 cell line should make it a useful addition to the cell lines currently available for in vitro and in vivo studies of glioblastoma multiforme.

Aged↗

PubMed1998

Pre-clinical evaluation of probes to detect t(8;21) AML minimal residual disease by fluorescence in situ hybridization.

The 8;21 translocation in acute myeloid leukemia (AML) results in a consistent fusion transcript, AML1/ETO. Long-term clinical remission occurs in some patients despite incomplete eradication of AML1/ETO as demonstrated by RT-PCR, thus limiting the usefulness of this assay. An important future goal will be to determine if there is a level of minimal residual disease (MRD) in patients below which relapse is unlikely. For the detection of MRD, we have developed reagents for fluorescence in situ hybridization (FISH) that identify both derivative 8 and 21 chromosomes with a high analytical sensitivity. In t(8;21) AML cells, two fused signals were detected in addition to the normal 8 and 21 alleles. The sensitivity and specificity of this probe mixture were analyzed in cell lines and patient bone marrows. One and two randomly juxtaposed signals were observed in 2.4 and 0.04% of normal cells, respectively. However, these were easily differentiated from t(8;21) cells by the absence of signals from the normal alleles. Using as criteria the presence of two fused signals plus the normal alleles, we observed no false positives among 5,000 normal cells. The probe correctly identified 20/20 patients with t(8;21) AML and 10/10 non-t(8;21) patients. In cell dilution experiments, the analytical sensitivity of this reagent was equal to that of the X chromosome and Y chromosome alpha-satellite probes. These optimized probes should facilitate the quantitative assessment and study of MRD in t(8;21) AML.

Bone Marrow Cells↗

PubMed1997

Homozygous deletions of human chromosome 3p in lung tumors.

Cytogenetic and loss of heterozygosity (LOH) studies have demonstrated that deletions of chromosome 3p occur at a high frequency in all forms of lung cancer. To clarify the role of 3p in lung tumorigenesis and to more precisely identify targets for positional cloning efforts, we have performed 3p deletion analyses (microsatellite and fluorescence in situ hybridization) in a series of lung cancer cell lines and uncultured tumor samples. Importantly, we identified homozygous deletions in four uncultured tumors and one cell line. Homozygous deletions were found in three squamous tumors within a region of 3p21 which had previously been described only in cell lines, a 1-2-megabase homozygous deletion in a small cell tumor at 3p12, and a 3p14.2 homozygous deletion in a non-small cell lung carcinoma cell line. The detection of homozygous deletions affecting these multiple regions in uncultured tumor cells substantiates the belief (previously based on deletions found only in tumor cell lines) that these sites contain important tumor suppressor genes. Along with previously reported homozygous deletions in a distal portion of 3p21.3, we now have evidence for four separate regions of 3p which undergo homozygous deletions in either uncultured lung tumors or cell lines.

Carcinoma, Small Cell↗

PubMed1997

Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.

Cytogenetic and molecular studies have implied the presence of tumor suppressor genes (TSGs) on chromosome 9p that are critical in the development of lung and other cancers. The p16/CDKN2 gene, a cyclin dependent kinase inhibitor, is a well-defined TSG on 9p21. Although the frequency of mutations in the p16/CDKN2 gene has been detected in approximately 30% of non-small cell lung cancer, loss of heterozygosity on 9p has been observed in greater than 70% of non-small cell lung cancers. These and other deletion mapping studies have suggested the existence of additional TSGs on 9p. This study examined chromosome 9p for TSG loci by analyzing 23 squamous cell carcinomas of the lung with 21 microsatellite markers. Loss of heterozygosity was detected in all of the tumors, and homozygous deletions of the p16/ CDKN2 locus were observed in 6 of the 23 tumors (26%). In addition, a novel region of homozygous deletion was detected in six tumors (26%) at D9S126, approximately 2.5 cM proximal to p16/CDKN2. A single tumor contained a homozygous deletion at both the p16/CDKN2 locus and the D9S126 locus. The possibility of homozygous loss was confirmed by multiplex PCR using both the D9S126 marker and a chromosome 9p control marker. Fluorescence in situ hybridization analysis with P1 and cosmid probes containing D9S126 also confirmed these data. The minimum region of homozygous deletion was determined by testing markers immediately proximal and distal to the D9S126 region. The data identify a homozygous loss on the short arm of chromosome 9 suggesting the presence of a novel TSG locus, proximal to p16/CDKN2 and located between D9S265 and D9S259.

Carcinoma, Squamous Cell↗

PubMed1997

The benzene metabolite, hydroquinone, induces dose-dependent hypoploidy in a human cell line.

Chronic exposure to high concentrations of benzene can result in the development of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving high frequency of loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8. The pattern of reoccurring chromosome abnormalities associated with the development of leukemia can be used as a guide in understanding the etiology and pathogenesis of these diseases. Therefore, a research project was designed to determine whether a metabolite of benzene, hydroquinone (HQ), could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7 and 8 probes we demonstrate that 42, 49 and 26 microM HQ induces monosomy 5, 7 and 8, respectively, in the human lymphoblast cell line GM09948. These results demonstrate for the first time that HQ induces a specific chromosome loss found in secondary MDS/AML. The pattern of chromosome 5 and/or 7 loss in benzene-induced MDS/AML is probably due to selective cell survival after HQ exposure rather than specific targeting of HQ for chromosomes 5 or 7.

Cells, Cultured↗