Biomedical subjects

Alexei Protopopov

Publications and source records attributed to Alexei Protopopov.

14 recordsLinked to original sources

PubMed2006

Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.

Animals↗

PubMed2006

Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations.

Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.

Animals↗

PubMed2006

High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients.

To identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

Chromosomes, Human↗

PubMed2005

Epigenetic transdifferentiation of normal melanocytes by a metastatic melanoma microenvironment.

The clinical management of cutaneous melanoma would benefit significantly from a better understanding of the molecular changes that occur during melanocytic progression to a melanoma phenotype. To gain unique insights into this process, we developed a three-dimensional in vitro model that allows observations of normal human melanocytes interacting with a metastatic melanoma matrix to determine whether these normal cells could be reprogrammed by inductive cues in the tumor cell microenvironment. The results show the epigenetic transdifferentiation of the normal melanocytic phenotype to that of an aggressive melanoma-like cell with commensurate increased migratory and invasive ability with no detectable genomic alterations. Removal of the transdifferentiated melanocytes from the inductive metastatic melanoma microenvironment results in a reversion to their normal phenotype. However, a normal melanocyte microenvironment had no epigenetic influence on the phenotype of metastatic melanoma cells. This novel approach identifies specific genes involved in the transdifferentiation of melanocytes to a more aggressive phenotype, which may offer significant therapeutic value.

Cell Differentiation↗

PubMed2005

hUNC-93B1, a novel gene mainly expressed in the heart, is related to left ventricular diastolic function, heart failure morbidity and mortality in elderly men.

AIMS: The hUNC-93B1 gene has the highest expression in the heart. We aimed to explore relationships between the hUNC-93B1 gene and cardiac function, morbidity and mortality in elderly men. METHODS AND RESULTS: Two sub-samples of the population-based ULSAM-cohort (n=330, mean age 71 years and n=152, mean age 75 years, respectively) were used to explore and validate relationships between genotypes of the hUNC-93B1 gene and cardiac phenotypes (ejection fraction, E/A-ratio, left ventricular mass index and relative wall thickness). In the two samples, subjects homozygous for haplotype H3 had 34% and 35% higher level of E/A-ratio compared to non-carriers (p=0.0002 and 0.017, respectively) independent of cardiovascular disease and medication. Using national cause-of-death and hospital-discharge register data with 29 years of follow-up, no heart failure patients homozygous for haplotype H3 were hospitalised for heart failure before the age of 75 years, compared to 25% for heterozygous and 55% for non-carriers (p<0.03). No homozygous subjects died during follow-up while 17% of the heterozygous and 15% of the non-carriers died (p=0.01). CONCLUSION: Haplotype H3 of the hUNC-93B1 gene seems related to E/A-ratio in elderly men. The relationship between the hUNC-93B1 gene and the age at onset of heart failure and mortality support a view of a clinically relevant impact of the gene.

Aged↗

PubMed2005

Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential.

BACKGROUND: In cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells. RESULTS: We designed siRNAs against two different regions of telomerase gene and evaluated their effect on telomere length, proliferative potential, and gene expression in Barrett's adenocarcinoma SEG-1 cells. The mixture of siRNAs in nanomolar concentrations caused a loss of telomerase activity that appeared as early as day 1 and was essentially complete at day 3. Inhibition of telomerase activity was associated with marked reduction in median telomere length and complete loss of detectable telomeres in more than 50% of the treated cells. Telomere loss caused senescence in 40% and apoptosis in 86% of the treated cells. These responses appeared to be associated with activation of DNA sensor HR23B and subsequent activation of p53 homolog p73 and p63 and E2F1. Changes in these gene regulators were probably the source of observed up-regulation of cell cycle inhibitors, p16 and GADD45. Elevated transcript levels of FasL, Fas and caspase 8 that activate death receptors and CARD 9 that interacts with Bcl10 and NFKB to enhance mitochondrial translocation and activation of caspase 9 were also observed. CONCLUSION: These studies show that telomerase siRNAs can cause effective suppression of telomerase and telomere shortening leading to both cell cycle arrest and apoptosis via mechanisms that include up-regulation of several genes involved in cell cycle arrest and apoptosis. Telomerase siRNAs may therefore be strong candidates for highly selective therapy for chemoprevention and treatment of Barrett's adenocarcinoma.

Adenocarcinoma↗

PubMed2005

High-resolution genomic profiles of human lung cancer.

Lung cancer is the leading cause of cancer mortality worldwide, yet there exists a limited view of the genetic lesions driving this disease. In this study, an integrated high-resolution survey of regional amplifications and deletions, coupled with gene-expression profiling of non-small-cell lung cancer subtypes, adenocarcinoma and squamous-cell carcinoma (SCC), identified 93 focal copy-number alterations, of which 21 span <0.5 megabases and contain a median of five genes. Whereas all known lung cancer genes/loci are contained in the dataset, most of these recurrent copy-number alterations are previously uncharacterized and include high-amplitude amplifications and homozygous deletions. Notably, despite their distinct histopathological phenotypes, adenocarcinoma and SCC genomic profiles showed a nearly complete overlap, with only one clear SCC-specific amplicon. Among the few genes residing within this amplicon and showing consistent overexpression in SCC is p63, a known regulator of squamous-cell differentiation. Furthermore, intersection with the published pancreatic cancer comparative genomic hybridization dataset yielded, among others, two focal amplicons on 8p12 and 20q11 common to both cancer types. Integrated DNA-RNA analyses identified WHSC1L1 and TPX2 as two candidates likely targeted for amplification in both pancreatic ductal adenocarcinoma and non-small-cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗

PubMed2004

High-resolution global profiling of genomic alterations with long oligonucleotide microarray.

Cancer represents the phenotypic end point of multiple genetic lesions that endow cells with a full range of biological properties required for tumorigenesis. Among the hallmark features of the cancer genome are recurrent regional gains and losses that, upon detailed characterization, have provided highly productive discovery paths for new oncogenes and tumor suppressor genes. In this study, we describe the use of an oligonucleotide-based microarray platform and development of requisite assay conditions and bioinformatic mining tools that permits high-resolution genome-wide array-comparative genome hybridization profiling of human and mouse tumors. Using a commercially available 60-mer oligonucleotide microarray, we demonstrate that this platform provides sufficient sensitivity to detect single-copy difference in gene dosage of full complexity genomic DNA while offering high resolution. The commercial availability of the microarrays and associated reagents, along with the technical protocols and analytical tools described in this report, should provide investigators with the immediate capacity to perform DNA analysis of normal and diseased genomes in a global and detailed manner.

Animals↗

PubMed2003

mSin3-associated protein, mSds3, is essential for pericentric heterochromatin formation and chromosome segregation in mammalian cells.

The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division. In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation. However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation. Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis. mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes. Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes. On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells.

Acetylation↗

PubMed2003

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.

Bacteria↗

PubMed2003

Deletion mapping using quantitative real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas.

We report chromosome 3p deletion mapping of 32 cervical carcinoma (CC) biopsies using 26 microsatellite markers located in frequently deleted 3p regions to detect loss of heterozygosity and homozygous loss. In addition, two STS markers (NLJ-003 and NL3-001) located in the 3p21.3 telomeric (3p21.3T) and 3p21.3 centromeric (3p21.3C) regions, respectively, were used for quantitative real-time PCR as TaqMan probes. We show that quantitative real-time PCR is reliable and sensitive and allows discriminating between 0, 1 and 2 marker copies per human genome. For the first time, frequent (five of 32 cases, i.e. 15.6%) homozygous deletions were demonstrated in CCs in both 3p21.3T and 3p21.3C regions. The smallest region homozygously deleted in 3p21.3C was located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene) and contains 17 genes previously defined as lung cancer candidate Tumor suppressor genes (TSG(s)). The smallest region homozygously deleted in 3p21.3T was flanked by D3S1298 and NL1-024 (D3S4285), excluding DLEC1 and MYD88 as candidate TSGs involved in cervical carcinogenesis. Overall, this region contains five potential candidates, namely GOLGA4, APRG1, ITGA9, HYA22 and VILL, which need to be analysed. The data showed that aberrations of either NLJ-003 or NL3-001 were detected in 29 cases (90.6%) and most likely have a synergistic effect (P<0.01). The study also demonstrated that aberrations in 3p21.3 were complex and in addition to deletions, may involve gene amplification as well. The results strongly suggest that 3p21.3T and 3p21.3C regions harbor genes involved in the origin and/or development of CCs and imply that those genes might be multiple TSG(s).

Carcinoma↗

PubMed2003

An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.

To facilitate the identification of tumor suppressor genes in the chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb physical and gene map of this segment (between markers D3S4285 and D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of the GB4 genetic map. We used NotI-linking and -jumping clones, sequence-tagged site PCR marker analysis, and multicolor and fiber fluorescence in situ hybridization to confirm the sequence order and map orientation. An integrated clone contig composed of 5 yeast artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artificial chromosome, and 8 NotI-linking clones provided the physical base of the map. We unequivocally established the order of 28 sequence-tagged sites and 35 genes in the region. Gaps between published bacterial artificial chromosome contigs were determined and covered by our own sequence data. Furthermore, three new genes were isolated, namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible protein, STI2, and the AP20-region gene 1, APRG1. The tumor suppressor gene candidate APRG1 was positioned close to the border of the homozygous deletion in a small cell lung cancer cell line ACC-LC5. Expression analysis with a tissue-specific panel of cDNA revealed seven distinct tissue-specific splice variants (A-G) of the message (size range, 1.0-1.8 kb). Although the gene was expressed at a low level in all tested tissues, comparatively higher expression was detected in pancreas (splice forms B and D), kidney (A) and placenta (B and C). The APRG1 gene encoded a predicted protein of 170 amino acids (isoform B), which had an NH2-terminal part conserved among members of the eukaryotic translation factor 6 gene family. A Prosite pattern corresponding to the cell attachment sequence Arg-Gly-Asp was also found. The presence of this domain raised the intriguing possibility that APRG1B may be directly involved in membrane interactions and cell adhesion. We showed that the AP20 region was duplicated during mammalian evolution and homologous gene clusters were present in human chromosome 2 and syntenic mouse regions on chromosomes 1, 2, and 9. Interestingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was located at the borders of both breakpoints, evolutionarily conserved gene cluster and homozygous deletions detected in lung, kidney and other cancers. NotI digestion revealed that the AP20 region was frequently and extensively methylated in renal carcinoma cell lines and tumor biopsies.

Animals↗

PubMed2002

NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes.

Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of NotI clone microarrays and genomic subtraction, because NotI recognition sites are closely associated with CpG islands and genes. We show here that the CODE (Cloning Of DEleted sequences) genomic subtraction procedure can be adapted to NotI flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A NotI-CODE procedure can be used to prepare NotI representations (NRs) containing 0.1-0.5% of the total DNA. The NRs contain, on average, 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. NotI microarrays offer a powerful tool with which to study carcinogenesis.

Animals↗

PubMed2002

The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells.

We analyzed expression status of the recently identified tumor suppressor geneRASSF1A in primary prostate carcinomas and in prostate cell lines. We found complete methylation of the RASSF1A promoter in 63% of primary microdissected prostate carcinomas (7 of 11 samples). The remaining 4 samples (37%) were partially methylated, possibly because of contamination with normal cells. No promoter methylation was observed in matching normal prostate tissues. High levels of RASSF1A transcript and no methylation of RASSF1A promoter were found in explanted primary normal prostate epithelial and stromal cells. Complete silencing and methylation of RASSF1A promoter was observed in five widely used prostate carcinoma cell lines, which acquired the ability to grow in culture spontaneously, including LNCaP, PC-3, ND-1, DU-145, 22Rv1, and one primary prostate carcinoma immortalized by overexpression of the human telomerase catalytic subunit (RC-58T/hTERT). However, no silencing of RASSF1A was found in four other prostate carcinoma cell lines, which were adapted for cell culture after transformation with human papillomaviral DNA. Suppression of cell growth in vitro was demonstrated after the reintroduction of RASSF1A-expressing construct into LNCaP prostate carcinoma cells. Our data implicate the RASSF1A gene in human prostate tumorigenesis.

Cell Division↗