Biomedical subjects

Michael R Rossi

Publications and source records attributed to Michael R Rossi.

13 recordsLinked to original sources

PubMed2007

Overlay analysis of the oligonucleotide array gene expression profiles and copy number abnormalities as determined by array comparative genomic hybridization in medulloblastomas.

Combined analysis of gene expression array data and array-based comparative genomic hybridization data have been used in a series of 26 pediatric brain tumors to define up- and downregulated genes that coincide with losses, gains, and amplifications involving specific chromosome regions. Frequent losses were defined in chromosome arms 3q, 6q, 8p, 10q, 16q, 17p, and gains were identified in chromosome 7, and chromosome arms 9p and 17q. Amplification of a 2p region was seen in only one tumor, which corresponded to increased expression of the MYCN and DDX1 genes. To facilitate the analysis of the two data sets, we have developed a custom overlay tool that defines genes that are underexpressed in regions of deletions and overexpressed in regions of gain, across the genome and specifically within regions showing recurrent involvement in medulloblastomas.

Cerebellar Neoplasms↗

PubMed2006

An efficient numerical technique for bioheat simulations and its application to computerized cryosurgery planning.

As a part of ongoing efforts to develop computerized planning tools for cryosurgery, the current study focuses on developing an efficient numerical technique for bioheat transfer simulations. Our long-term goal is to develop a planning tool for cryosurgery that takes a 3D reconstruction of a target region, and suggests the best cryoprobe layout. Toward that goal, a planning algorithm, termed "force-field analogy," has been recently presented, based on a sequence of bioheat transfer simulations, which are by far the most computationally expensive part of the planning method. The objective in the current study is to develop a finite difference numerical scheme for bioheat transfer simulations, which reduces the overall run time of computerized planning, thereby making it clinically relevant. While the general concept of variable grid size and time intervals is not new, its application to the phase change problem of cryosurgery is the unique contribution of the current study.

Computer Simulation↗

PubMed2006

Array CGH analysis of pediatric medulloblastomas.

Brain tumors are the second most common childhood cancer. We used high-resolution array comparative genomic hybridization (aCGH) to analyze losses and gains of genetic material from 24 medulloblastomas. The bacterial artificial chromosome clones were ordered on the array, allowing for an average resolution of approximately 420 kilobases. The advantage of this high resolution is that the breakpoints associated with subregional chromosome copy number aberrations can be accurately defined, which in turn allows candidate genes within these regions to be readily defined. In this analysis, we confirmed the frequent involvement of loss of 17p and gain of 17q, although we have now established the position of the breakpoint that consistently lies in the chr17:18318880-19046234 region of the chromosome. Other frequent losses were seen on 8p, 10q, 16q, and 20p, and frequent gains were seen on 2p, 4p, 7, and 19. In addition, the fine-resolution mapping provided by aCGH made it possible to define small chromosome deletions in 1q23.3-q24.2, 2q13.12-q13.2, 6q25-qter, 8p23.1, 10q25.1, and 12q13.12-q13.2. Overall, amplification events were rare, the most common involving MYC (16%), on 8q, although isolated events were seen in 10p11 and 3q.

Cerebellar Neoplasms↗

PubMed2005

Molecular study of malignant gliomas treated with epidermal growth factor receptor inhibitors: tissue analysis from North American Brain Tumor Consortium Trials 01-03 and 00-01.

PURPOSE: We investigated the molecular effect of the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib in vivo on all available tumors from patients treated on North American Brain Tumor Consortium trials 01-03 and 00-01 for recurrent or progressive malignant glioma. EXPERIMENTAL DESIGN: EGFR expression and signaling during treatment with erlotinib or gefitinib were analyzed by Western blot and compared with pre-erlotinib/gefitinib-exposed tissue or unexposed controls. Tumors were also analyzed for EGFR mutations and for other genomic abnormalities by array-based comparative genomic hybridization. Clinical data were used to associate molecular features with tumor sensitivity to erlotinib or gefitinib. RESULTS: Erlotinib and gefitinib did not markedly affect EGFR activity in vivo. No lung signature mutations of EGFR exons 18 to 21 were observed. There was no clear association between erlotinib/gefitinib sensitivity and deletion or amplification events on array-based comparative genomic hybridization analysis, although novel genomic changes were identified. CONCLUSIONS: As erlotinib and gefitinib were generally ineffective at markedly inhibiting EGFR phosphorylation in these tumors, other assays may be needed to detect molecular effects. Additionally, the mechanism of erlotinib/gefitinib sensitivity likely differs between brain and lung tumors. Finally, novel genomic changes, including deletions of chromosomes 6, 21, and 22, represent new targets for further research.

Antineoplastic Agents↗

PubMed2005

Differential expression of the LGI and SLIT families of genes in human cancer cells.

The LGI and SLIT genes have a distinctive leucine-rich repeat motif in the N-terminal end of the protein which is indicative of either receptor function or an interaction with the extracellular matrix. Members of the LGI and SLIT family of genes have been implicated in specific cancers and have been suggested to have a restricted pattern of expression in normal cells. To investigate the extent and distribution of the expression of these genes in cancer cells we have analyzed their expression levels in a range of tumor cell types. Different tumor types appear to hold a preference for the specific members of the families which are expressed. Differential expression between cell lines, from the same tumor type, implies a role for inactivation and reactivation of these genes during tumorigenesis. The detailed characterization of the expression pattern in these tumor cells offers the opportunity to perform a functional analysis of these individual genes.

Breast Neoplasms↗

PubMed2005

Identification of consistent novel submegabase deletions in low-grade oligodendrogliomas using array-based comparative genomic hybridization.

We have analyzed 18 low-grade gliomas using array comparative genomic hybridization (aCGH) with an average resolution of <500 kb. Because the majority of these tumors showed loss of chromosome arms 1p and 19q, we used custom statistical approaches to define submegabase hemizygous losses throughout the genome that correlated with 19q loss. As a result of this analysis, we have identified a approximately 550-kb region in 11q13 and a approximately 300-kb region in 13q12 that showed hemizygous deletion in virtually all the tumors analyzed regardless of their 1p/19q status. FISH analyses of interphase nuclei from the same tumors used for aCGH analysis confirmed the hemizygous loss. The identification of such specific changes provides a potentially very useful diagnostic marker for this subgroup of low-grade tumors. These regions of the genome define small numbers of candidate genes that are within the deletions. The aCGH analysis also defined the spectrum of gain and loss of genomic regions in low-grade oligodendrogliomas.

Brain Neoplasms↗

PubMed2005

Novel amplicons on the short arm of chromosome 7 identified using high resolution array CGH contain over expressed genes in addition to EGFR in glioblastoma multiforme.

Amplification of a defined chromosome segment on the short arm of chromosome 7 has frequently been reported in glioblastoma multiforme (GBM), where it is generally assumed that it is the result of over expression of the epidermal growth factor receptor (EGFR) gene that provides the selective pressure to maintain the amplification event. We have used high resolution array comparative genomic hybridization (aCGH) to analyze amplification events on chromosome 7p in GBM, which demonstrates that, in fact, several other regions distinct from EGFR can be amplified. To determine the changes in gene expression levels associated with these amplification events, we used oligonucleotide expression arrays to investigate which of the genes in the amplified regions were also over expressed. These analyses demonstrated that not all genes in the amplicons showed increased expression, and we have defined a series of over expressed genes on 7p that could potentially contribute to the development of the malignant phenotype in these tumors. The global analysis of amplification afforded by aCGH analysis has improved our ability to define numerical chromosome abnormalities in cancer cells and has raised the possibility that genes other than EGFR may be important.

Chromosomes, Artificial, Bacterial↗

PubMed2005

Molecular characterization of a consistent 4.5-megabase deletion at 4q28 in prostate cancer cells.

Spectral karyotyping of prostate cell lines LNCaP, DU145, PC3, and 22RV demonstrated structural chromosome rearrangements involving the distal long arm of chromosome 4. In all but 22RV, these are nonreciprocal translocations between chromosomes 4 and 10. In 22RV, an apparently reciprocal t(2q;4q) is seen. Fluorescence in situ hybridization analysis of the chromosome 4 translocation breakpoints demonstrated that deletions were associated with all of the translocations, resulting in a net loss of chromosome material. Overlapping deletions in 4q28 approximately 34 were seen in LNCap, DU145, and 22RV, which defined an approximately 4.5-megabase pair common region of deletion. The deletion in PC3 was more proximal on 4q, involving the 4q21 approximately q26 region. A meta analysis of high-resolution definition of losses of chromosome material from published studies demonstrates that loss of 4q material may occur in at least 50% of primary tumors. This analysis defines a series of genes in the critical 4q region, which is potentially associated with prostate tumor development.

Chromosome Deletion↗

PubMed2005

Identification of inactivating mutations in the JAK1, SYNJ2, and CLPTM1 genes in prostate cancer cells using inhibition of nonsense-mediated decay and microarray analysis.

We have developed a simple analytical method that increases the efficiency of identifying mutant genes in cell lines after the inhibition of nonsense-mediated decay (NMD). The approach assumes that the spectra of mutant genes differ between cell lines of the same tumor origin. Thus, by analyzing more than one cell line in parallel and taking into account not only changes in mRNA levels after the inhibition of NMD, but also comparing mRNA levels between cell lines before the inhibition of NMD, the vast majority of false positives were eliminated from the analysis. In this study, we used Affymetrix oligonucleotide arrays to compare mRNA profiles of two prostate cancer cell lines, PC3 and LNCaP, before and after emetine treatment. As a result of our modified approach, from the 14,500 genes present on the array, 7 were identified as candidates from LNCaP cells and 1 was identified from PC3 cells. Sequence analysis of five of these candidate genes identified gene-inactivating mutations in four of them. Homozygous mutations were found in the synaptojanin 2 (SYNJ2) and the cleft lip and palate CLPTM1 genes. Two different heterozygous mutations in the Janus kinase 1 (JAK1) gene result in complete loss of the protein in several different prostate cancer cell lines.

Codon, Nonsense↗

PubMed2005

Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line.

The t(3;9)(p14;p21) in the MCF10A human mammary gland epithelial cell line was the single cytogenetic event that accompanied the transition from primary culture to immortalized cell line, suggesting that it is related to the development of the immortalization phenotype. To study the molecular consequences of the breakpoints in this rearrangement, we used a combination of fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (CGH). The 3p14 translocation breakpoint occurs within BAC RP11-795e22, which accommodates only the TAFA1 gene, a novel cysteine-rich secreted protein thought to be involved in cytokine signaling. TAFA1 is expressed in normal breast tissue, not in MCF10A, and shows differential expression in a range of breast cancer cell lines. The 9p translocation breakpoint results in a deletion of approximately 4 megabases on the derivative chromosome 9, which includes the CDKN2A (p16) gene. Array CGH and FISH analysis demonstrated that BAC 149i22, which contains the CDKN2A/B genes, is also deleted specifically on the apparently normal copy of chromosome 9, making MCF10A null for the p16/p15 genes. The exact extent of gains and losses of chromosome regions resulting from rearrangements involving chromosomes 1q, 5q, and 8q have also been characterized using the BAC arrays.

Cell Line, Tumor↗

PubMed2005

Truncating mutations in the ACVR2 gene attenuates activin signaling in prostate cancer cells.

Activins are classified as members of the TGFbeta superfamily of signaling molecules and both activin and TGFbeta ligands signal through structurally and functionally related serine/threonine kinase receptors. Defects in these signaling pathways have been associated with the initiation and progression of the cancer phenotype. Inactivating mutations in the TGFbeta type II receptor gene, TGFbetaR2, have been identified in a variety of tumors and cell lines, particularly those with microsatellite instability (MSI). More recently, mutations in the activin type II receptor gene, ACVR2, were identified in colon and pancreatic cell lines and tumors with MSI. Because prostate tumors appear to have a high incidence of MSI, we analyzed prostate cancer cell lines, with and without MSI, for ACVR2 and TGFbetaR2 mutations. Our analysis of 6 prostate cell lines revealed mutations in the ACVR2 gene in 22Rv-1, LAPC-4, DU145, and LNCaP cells and mutations in the TGFbetaR2 gene in 22Rv-1 and LAPC-4. PC3 and H660 cells were wild-type for ACVR2 and TFGbetaR2. All of the ACVR2 mutations were truncating mutations, and using an activin response assay, we demonstrate that truncating mutations of the ACVR2 gene result in a significant reduction in activin mediated cell signaling. Inactivation of ACVR2 is a common event in prostate cancer cells suggesting it may play an important role in the development of prostate cancer.

Activin Receptors↗

PubMed2003

Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids.

Mouse-human somatic cell hybrids have been extensively used in the molecular genetic dissection of human disease-related chromosome rearrangements because of their ability to selectively and randomly eliminate human chromosomes. This technology allows the isolation of structural chromosome abnormalities, which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions and translocations, down to the nucleotides involved. The main confounding problem with the analysis of somatic cell hybrids is determining the exact chromosome complement unequivocally and quickly. Spectral karyotyping can identify each of the individual human chromosomes in a normal metaphase spread, as well as structural chromosome rearrangements-although, because of potential cross-hybridization between the human probe and mouse DNA sequences during the hybridization reaction, it has not been determined whether the same analysis will selectively identify human chromosomes on a mouse background. We show (to our knowledge, for the first time) that, under modified conditions of chromosomal in situ suppression hybridization, the standard spectral karyotyping probe does not cross-react with mouse chromosomes and can be used to identify subtle structurally rearranged chromosomes in hybrid cells. This analysis allows for the rapid and unequivocal identification of the human chromosome complement in these hybrids, as well as structural chromosome rearrangements that occur between mouse and human chromosomes that might otherwise confound the analysis.

3T3 Cells↗

PubMed2002

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 stress response genes in cultured human urothelial cells (UROtsa) exposed to lethal and sublethal concentrations of sodium arsenite.

The stress response is one mechanism that the bladder urothelium could potentially employ to protect itself from cellular damage after exposure to arsenic and, in so doing, influence the shape of the dose-response curve at low concentrations of exposure to this environmental pollutant. In the present study, we used the cultured human urothelial cell line UROtsa, a model of human urothelium, to determine the expression of heat shock proteins hsp 27, hsp 60, hsc 70, and hsp 70 after acute and extended exposure of the cells to lethal and sublethal levels of sodium arsenite (NaAsO2). Acute exposure was modeled by exposing confluent cultures of UROtsa cells to 100 micro M NaAsO2 for 4 hr followed by a 48-hr recovery period. Extended exposure was modeled by exposing confluent UROtsa cells to 1, 4, and 8 micro M NaAsO2 for 16 days, with the highest concentration producing cell death by 4 days of exposure. The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined by reverse-transcription polymerase chain reaction and Western analysis. Cell viability was determined by the MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results demonstrated that the expression of hsp 27, hsp 60, and hsc 70 mRNA and protein were not consistently increased by either acute or extended exposure to NaAsO2. In contrast, hsp 70 expression was induced by NaAsO2 after both acute and extended exposure. The degree and duration of the induction of the hsp 70 protein in the extended time course of exposure to NaAsO2 correlated directly with UROtsa cell cytotoxicity. The substantial level of basal expression of hsp 27, hsp 60, and hsc 70 shown previously in human bladder urothelium, coupled with the inducible expression of hsp 70, could provide the human urothelium with a mechanism to withstand and recover from a low level of arsenite exposure.

Arsenates↗