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Biomedical subjects

N Popescu

Publications and source records attributed to N Popescu.

At least 19 recordsLinked to original sources
PubMed2001

Derivation of human tumor cells in vitro without widespread genomic instability.

The majority of adult human epithelial cancers exhibit evidence of genetic instability, and it is widely believed that the genetic instability manifested by aneuploidy or microsatellite instability plays an essential role in the genesis of these tumors. Indeed, most experimental models of cancer also show evidence of genomic instability. The resulting genetic chaos, which has widespread effects on many genes throughout the genome, confounds attempts to determine the precise cohort of genetic changes that are required for the transformation of normal human cells to a tumorigenic state. Here we show that genetic transformation of human kidney epithelial cells can occur in the absence of extensive aneuploidy, chromosomal translocations, and microsatellite instability. These observations demonstrate that the in vitro oncogenic transformation of human cells can proceed without widespread genomic instability.

Cell Line, Transformed↗
PubMed2001

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Animals↗
PubMed2001

Molecular characterization of murine pancreatic phospholipase A(2).

The pancreatic secretory phospholipase A(2) (sPLA(2)IB) is considered to be a digestive enzyme, although it has several important receptor-mediated functions. In this study, using the newly isolated murine sPLA(2)IB cDNA clone as a probe, we demonstrate that in addition to the pancreas, the sPLA(2)IB mRNA was expressed in extrapancreatic organs such as the liver, spleen, duodenum, colon, and lungs. We also demonstrate that sPLA(2)IB mRNA expression was detectable from the 17(th) day of gestation in the developing mouse fetus, coinciding with the time of completion of differentiation of the pancreas. Furthermore, the mRNA expression pattern of sPLA(2)IB was distinct from those of sPLA(2)IIA and cPLA(2) in various tissues examined. The murine sPLA(2)IB gene structure is well conserved, consistent with findings in other mammalian species, and this gene mapped to the region of mouse chromosome 5F1-G1.1. Taken together, our results suggest that sPLA(2)IB plays important roles both in the pancreas and in extrapancreatic tissues and that in the mouse, its expression is developmentally regulated.

Amino Acid Sequence↗
PubMed2000

Identification of a YAC from 16q24 carrying a senescence gene for breast cancer cells.

We have identified a 360 kb YAC that carries a cell senescence gene, SEN16. In our earlier studies, we localized SEN16 within a genetic interval of 3 - 7 cM at 16q24.3. Six overlapping YACs spanning the chromosomal region of senescence activity, were assembled in a contig. Candidate YACs, identified by the markers located in the vicinity of SEN16, were retrofitted to introduce a neo selectable marker. Retrofitted YACs were first transferred into mouse A9 cells to generate A9/YAC hybrids. YAC DNA present in A9/YAC hybrids was further transferred by microcell fusion into immortal cell lines derived from human and rat mammary tumors. YAC d792t2 restored senescence in both human and rat mammary tumor cell lines, while an unrelated YAC from chromosome 6q had no senescence activity.

Aging↗
PubMed2000

Human acid ceramidase gene: novel mutations in Farber disease.

Farber disease is an autosomal recessive disorder caused by lysosomal acid ceramidase (AC) deficiency. It commonly manifests during the first few months after birth with a unique triad of painful and progressive deformed joints, subcutaneous nodules, and progressive hoarseness. In order to understand the molecular mechanism(s) of pathogenesis of Farber disease, we isolated and characterized a full-length human AC gene, mapped its chromosomal location, determined the tissue-specific expression, and analyzed mutations in Farber disease patients. We also studied the AC-mRNA expression in gastrointestinal tumors and adjoining normal tissues. In addition, we determined the pattern of tissue-specific AC-mRNA expression in the adult mouse and during fetal development. Our results show that human AC gene consists of 14 exons and 13 introns spanning approximately 26.5 kb of genomic DNA. It is mapped to human chromosome 8p22-21.2, a region often disrupted in several cancers. The AC-mRNA is expressed in the mouse fetus from the seventh day of gestation. Interestingly, while the AC-mRNA is expressed in all segments of the normal gastrointestinal tract, none of the gastrointestinal tumor tissues had any AC-mRNA expression. We also uncovered four novel mutations in Farber disease patients that were not previously reported. Taken together, our results not only attest to the physiological importance of AC but also uncover several new mutations in Farber disease that may advance our knowledge towards establishing a genotype-phenotype correlation in this disease.

Acid Ceramidase↗
PubMed1999

Defects in transforming growth factor-beta signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes.

Genetic inactivation of the transforming growth factor-beta (TGF-beta) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-ras(Ha) transduced primary TGF-beta1-/- keratinocytes and keratinocytes expressing a TGF-beta type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-beta1-/- keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-beta signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-beta1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-beta1-/- keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-beta signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Aneuploidy↗
PubMed1999

Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping.

We revisited the cytogenetic alterations of the cervical adenocarcinoma cell line HeLa through the use of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH). SKY analysis unequivocally characterized all abnormal chromosomes. Chromosomal breakpoints were primarily assigned by simultaneous assessment of SKY painted chromosomes and inverted 4,6-diamidino2-phenylindole banding from the same cell. Twenty clonally abnormal chromosomes were found. Comparison with previously reported HeLa G-banding karyotypes revealed a remarkably stable cytogenetic constitution because 18 of 20 markers that were found were present before. The classification of 12 markers was refined in this study. Our assignment of the remaining six markers was consistent with those described in the literature. The CGH map of chromosomal copy number gains and losses strikingly matched the SKY results and was, in a few instances, decisive for assigning breakpoints. The combined use of molecular cytogenetic methods SKY, CGH, and FISH with site-specific probes, in addition to inverted 4,6-diamidino-2-phenylindole or conventional G-banding analysis, provides the means to fully assess the genomic abnormalities in cancer cells. Human papillomaviruses (HPVs) are frequently integrated into the cellular DNA in cervical cancers. We mapped by FISH five HPV18 integration sites: three on normal chromosomes 8 at 8q24 and two on derivative chromosomes, der(5)t(5;22;8)(qll;q11q13;q24) and der(22)t(8; 22)(q24;q13), which have chromosome 8q24 material. An 8q24 copy number increase was detected by CGH. Dual-color FISH with a c-MYC probe mapping to 8q24 revealed colocalization with HPV18 at all integration sites, indicating that dispersion and amplification of the c-MYC gene sequences occurred after and was most likely triggered by the viral insertion at a single integration site. Numerical and structural chromosomal aberrations identified by SKY, genomic imbalances detected by CGH, as well as FISH localization of HPV18 integration at the c-MYC locus in HeLa cells are common and representative for advanced stage cervical cell carcinomas. The HeLa genome has been remarkably stable after years of continuous cultivation; therefore, the genetic alterations detected may have been present in the primary tumor and reflect events that are relevant to the development of cervical cancer.

Chromosome Aberrations↗
PubMed1998

Characterization and chromosomal localization of human hair-specific keratin genes and comparative expression during the hair growth cycle.

During anagen, cell proliferation in the germinative matrix of the hair follicle gives rise to the fiber and inner root sheath. The hair fiber is constructed from structural proteins belonging to four multigene families: keratin intermediate filaments, high-sulfur matrix proteins, ultra high-sulfur matrix proteins, and high glycine-tyrosine proteins. Several hair-specific keratin intermediate filament proteins have been characterized, and all have relatively cysteine-rich N- and C-terminal domains, a specialization that allows extensive disulfide cross-linking to matrix proteins. We have cloned two complete type II hair-specific keratin genes (ghHb1 and ghHb6). Both genes have nine exons and eight introns spanning about 7 kb and lying about 10 kb apart. The structure of both genes is highly conserved in the regions that encode the central rod domain but differs considerably in the C-terminal coding and noncoding sequences, although some conservation of introns does exist. These genes have been localized to the type II keratin cluster on chromosome 12q13 by fluorescence in situ hybridization. They, and their type I partner ghHa1, are expressed in differentiating hair cortical cells during anagen. In cultured follicles, ghHa1 expression declined in cortical cells and was no longer visible after 6 d, whereas the basal epidermal keratin hK14 appeared in the regressing matrix. The transition from anagen to telogen is marked by downregulation of hair cortical specific keratins and the appearance of hK14 in the epithelial sac to which the telogen hair fiber is anchored. Further studies of the regulation of these genes will improve our understanding of the cyclical molecular changes that occur as the hair follicle grows, regresses, and rests.

Base Sequence↗
PubMed1995

Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.

Human macrophage metalloelastase (HME) is a recent addition to the matrix metalloproteinase (MMP) family that was initially found to be expressed in alveolar macrophages of cigarette smokers. To understand more about HME expression, analysis of the structure and location of the gene was performed. The gene for HME is composed of 10 exons and 9 introns, similar to the stromelysins and collagenases, and HME shares the highly conserved exon size and intron-exon borders with other MMPs. The 13-kilobase (kb) HME gene has been localized by fluorescence in situ hybridization to chromosome 11q22.2-22.3, the same location of the interstitial collagenase and stromelysin genes. We determined that HME and stromelysin 1 genes are physically linked within 62 kb utilizing pulse-field gel electrophoresis. The promoter region of the HME gene contains several features common to other MMP genes including a TATA box 29 bp upstream to the transcription initiation site, an AP-1 motif, and a PEA3 element. HME mRNA is not detectable in normal adult tissues but is induced in rapidly remodeling tissues such as the term placenta. In situ hybridization and immunohistochemistry of placental tissue demonstrated HME mRNA and protein expression in macrophages and stromal cells. Cell-specific expression and response to inflammatory stimuli such as endotoxin is conferred within 2.8 kb of the HME 5'-flanking sequence as demonstrated by HME promoter-CAT expression constructs. Knowledge of the genomic organization and chromosomal location of HME may allow us to further define mechanisms responsible for cell- and tissue-specific expression of HME.

Base Sequence↗
PubMed1994

Chromosomal localization of the human histone H2A.X gene to 11q23.2-q23.3 by fluorescence in situ hybridization.

The human histone H2A.X gene is unusual in that its transcripts are alternatively processed to yield two species, one a 0.6-kb replication-linked histone mRNA and the other a 1.6-kb polyadenylated mRNA. The H2A.X gene has been localized by fluorescence in situ hybridization to chromosome 11q23.2-q23.3, away from the known clusters of human histone genes on chromosomes 1, 6, and 12. Assignment to chromosome 11 was substantiated by analysis of human-hamster somatic cell hybrid lines. As this work was being completed, an 89-bps sequence overlap was found between the downstream regions of the H2A.X gene and the recently sequenced hydroxymethylbilane (HMB)-synthase gene. The H2A.X and HMB-synthase genes have an unusual arrangement, being transcribed towards each other with their polyadenylation sites 330 bp apart. In addition the HMB-synthase gene contains constitutive and erythroid specific promoters. K562, an erythroid cell line, was found to contain a high concentration of the 1.6-kb polyadenylated H2A.X mRNA.

Animals↗
PubMed1994

Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression.

To examine whether human herpesvirus 6 (HHV-6) is capable of infecting human cervical epithelial cells and altering expression of human papillomavirus (HPV) genes, HPV-immortalized or -transformed carcinoma cell lines were infected with HHV-6 variant A. No cytopathic effect was observed in infected cervical cells. However, immunofluorescence indicated that infected cells expressed early-late proteins of HHV-6 by day 3 postinfection. HHV-6 DNA was also detected by Southern blot hybridization after infection and persisted through continued subculture in an episomal state as proven by Gardella gel electrophoresis and fluorescence in situ hybridization. HHV-6 infection enhanced expression of HPV RNAs encoding the viral oncoproteins E6 and E7. Transient transfection assays showed that two HHV-6 molecular clones, pZVB-70 and pZVH-14, upregulated transcription 9- to 15-fold from a receptor plasmid containing the HPV type 18 regulatory sequences which control transcription in vivo. Cervical carcinoma cells infected with HHV-6 induced more rapid development of tumors in mice than did noninfected cells. These results are the first evidence that human cervical epithelial cells can be infected with HHV-6 and that HHV-6 contains transactivators which stimulate the HPV-transforming genes.

Animals↗
PubMed1990

Mapping the human gene coding for chromosomal protein HMG-17.

The functional gene coding for nonhistone chromosomal protein HMG-17, a nucleosomal binding protein that may confer unique properties to the chromatin structure of active genes, has been mapped to band 1p36.1. The multiple, nonfunctional, HMG-17 retropseudogenes are scattered over several chromosomes.

Chromosome Mapping↗
PubMed1990

Chromosomal protein HMG-14 gene maps to the Down syndrome region of human chromosome 21 and is overexpressed in mouse trisomy 16.

The gene for human high-mobility-group (HMG) chromosomal protein HMG-14 is located in region 21q22.3, a region associated with the pathogenesis of Down syndrome, one of the most prevalent human birth defects. The expression of this gene is analyzed in mouse embryos that are trisomic in chromosome 16 and are considered to be an animal model for Down syndrome. RNA blot-hybridization analysis and detailed analysis of HMG-14 protein levels indicate that mouse trisomy 16 embryos have approximately 1.5 times more HMG-14 mRNA and protein than their normal littermates, suggesting a direct gene dosage effect. The HMG-14 gene may be an additional marker for the Down syndrome. Chromosomal protein HMG-14 is a nucleosomal binding protein that may confer distinct properties to the chromatin structure of transcriptionally active genes and therefore may be a contributing factor in the etiology of the syndrome.

Animals↗
PubMed1989

Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes.

A genomic sequence and cloned complementary DNA has been identified for a novel receptor-like gene of the PDGF receptor/CSF1 receptor subfamily (platelet-derived growth factor receptor/colony-stimulating factor type 1 receptor). The gene recognized a 6.4-kilobase transcript that was coexpressed in normal human tissues with the 5.3-kilobase PDGF receptor messenger RNA. Introduction of complementary DNA of the novel gene into COS-1 cells led to expression of proteins that were specifically detected with antiserum directed against a predicted peptide. When the new gene was transfected into COS-1 cells, a characteristic pattern of binding of the PDGF isoforms was observed, which was different from the pattern observed with the known PDGF receptor. Tyrosine phosphorylation of the receptor in response to the PDGF isoforms was also different from the known receptor. The new PDGF receptor gene was localized to chromosome 4q11-4q12. The existence of genes encoding two PDGF receptors that interact in a distinct manner with three different PDGF isoforms likely confers considerable regulatory flexibility in the functional responses to PDGF.

Amino Acid Sequence↗