Sinoula Apostolou

Growth inhibition and induction of apoptosis in mesothelioma cells by selenium and dependence on selenoprotein SEP15 genotype.

Malignant mesotheliomas (MMs) are aggressive tumors derived from mesothelial cells lining the lungs, pericardium and peritoneum, and are often associated with occupational asbestos exposure. Suppression subtractive hybridization was used to identify genes differentially expressed in MM cells compared to normal mesothelial cells. A gene, SEP15, encoding a 15-kDa selenium-containing protein was isolated using this approach and was subsequently shown to be downregulated in ∼60% of MM cell lines and tumor specimens. A SEP15 polymorphic variant, 1125A, resides in the SECIS recognition element in the 3′-UTR and may influence the efficiency of Sec incorporation into the protein during translation. Since previous studies have implicated a potential role of the trace element selenium as a chemopreventive agent in animal models and in several types of human cancer, we investigated the effect of selenium on MM cells and its dependence on SEP15 genotype. Selenium was shown to inhibit cell growth and induce apoptosis in a dose-dependent manner in MM cells but had minimal effect on normal mesothelial cells. However, MM cells with downregulated SEP15 or the 1125A variant were somewhat less responsive to the growth inhibitory and apoptotic effects of selenium than MM cells expressing wild-type protein. RNAi-based knockdown studies demonstrated that SEP15 inhibition makes sensitive MM cells more resistant to selenium. These data imply that selenium may be useful as a chemopreventive agent in individuals at high risk of MM due to asbestos exposure, although those with the 1125A polymorphism may be less responsive to the protective benefits of dietary selenium supplementation.

A Novel Cas Family Member, HEPL, Regulates FAK and Cell Spreading

For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.

High-Resolution Cytogenetic-Based Physical Map of Human Chromosome 16

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.

Evaluation of a cosmid contig physical map of human chromosome 16

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.

Absence of BCL10 mutations in human malignant mesothelioma.

It has recently been suggested that a novel gene, BCL10, is mutated in mucosa-associated lymphoid tissue (MALT) B cell lymphoma and multiple tumor types (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999). This gene was identified through the cloning of a (1;14)(p22;q32) translocation breakpoint in a case of low-grade MALT lymphoma. Mutations of BCL10 were identified not only in MALT lymphomas, but also in other lymphoid tumors of B or T cell lineage, and tumor cell lines derived from malignant mesotheliomas (MMs), germ cell neoplasms, and colon carcinomas. Thus, the investigators proposed that alterations of BCL10 may contribute to the pathogenesis of several types of neoplasia.The observation of most interest to us was that BCL10, which maps to chromosome 1p22, exhibited mutations in all three MM cell lines analyzed. We had previously shown 1p22 to be a frequent site of allelic loss in MM (Lee et al. 1996xLee, W.-C., Balsara, B., Liu, Z., Jhanwar, S.C., and Testa, J.R. Cancer Res. 1996; 56: 4297–4301PubMedSee all ReferencesLee et al. 1996), which implicated the involvement of a tumor suppressor gene (TSG) at this location. The aim of this letter is to report the results of our studies of BCL10 as a candidate for the 1p22 putative TSG in MM, and to prompt further discussion about the potential role of this gene in human malignancy.Fifty MM cell lines were examined for loss of heterozygosity (LOH). Since YAC 929E1 contains BCL10 (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999), two microsatellite markers, D1S2766 and D1S1618, were selected for analysis, as they were determined through a database search (http://www-genome.wi.mit.edu) and PCR analysis to map to this YAC. Overall, 25 of 45 informative cases (55.6%) showed LOH of at least one of these two loci, with the higher frequency of allelic loss being at marker D1S1618 (18 out of 30 informative cases, 60%) (data not shown). However, the percentage of MMs with LOH in the vicinity of BCL10 is less than that previously observed for markers within the minimally deleted 1p22 region in this malignancy (Lee et al. 1996xLee, W.-C., Balsara, B., Liu, Z., Jhanwar, S.C., and Testa, J.R. Cancer Res. 1996; 56: 4297–4301PubMedSee all ReferencesLee et al. 1996). Furthermore, YAC 929E1 resides distal to this minimally deleted segment.RT-PCR was performed on MM cell lines that displayed LOH to determine the expression pattern of BCL10. Normal mesothelial cells and MM cell lines not exhibiting LOH at 1p22 were included as controls. All cell lines examined expressed BCL10 at similar levels (data not shown). The housekeeping gene GAPDH was also expressed at uniform levels in all cell lines tested. In summary, BCL10 was demonstrated to map to a region showing frequent LOH in MM, but expression of BCL10 did not appear to be altered in any of the tumor cell lines examined.To determine whether the BCL10 gene is mutated in MM, SSCP analysis was performed on genomic DNAs from 25 MM cell lines displaying LOH in 1p22, using primers described by Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999. Peripheral blood lymphocyte (PBL) samples from two healthy donors were included as controls. Band shifts were observed in MM PCR products amplified with primers specific for exons 1 and 3.2 (257 bp at the 3′ end of exon 3) of BCL10. Variant exon 1 bands were demonstrated in 18 of 25 MM cell lines. However, these band shifts appeared to be identical in 17 of these cell lines and in one of the normal PBL samples. Products from two MM cell lines amplified with exon 3.2 primers displayed identical banding patterns, which differed from those observed in normal PBL samples. Examples of band shifts are shown in Figure 1Figure 1. We also carried out SSCP analysis of eight tumor samples matched to MM cell lines displaying variant bands. In each case, identical patterns were observed in the tumor and the corresponding MM cell line. Variant bands were not observed in BCL10 products from exons 2 and 3.1 (262 bp at the 5′ end of exon 3).Figure 1SSCP and Sequence Analyses of BCL10 in MM Cell LinesSSCP analysis was performed on genomic DNA. The entire coding sequence of BCL10 was amplified with AmpliTaq (Perkin Elmer) and previously described primers (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999), using standard PCR conditions. Autoradiographs at left show PCR products generated by primers corresponding to exon 3.2 (A) or exon 1 (B) analyzed on 10% nondenaturing acrylamide gels. Lanes 1–4: MM cell lines, as indicated; lane 5: PBL DNA from a normal individual (PBL). Templates for sequencing were amplified from genomic DNA by PCR with AmpliTaq and primers flanking each exon of BCL10. The chromatograms at right show sequence differences between Meso 17 and PBL (A) or between Meso 38 and PBL (B). Arrows indicate altered nucleotides.View Large Image | View Hi-Res Image | Download PowerPoint SlideReproducible band shifts in DNA from MM cases were further investigated by direct sequencing of purified PCR products. The sequences derived from the MM samples were compared with the published BCL10 sequence (accession number AJ006288). Nucleotide alterations detected in PCR products from MM samples are presented in Table 1Table 1. Some of these changes were predicted to result in substitutions of amino acids at the corresponding positions in the protein sequence. In each of these cases, individual tumor DNA samples possessed the same sequence as that of the corresponding cell line.Table 1DNA Sequence Alteration of BCL10 Detected in MMMeso Cell LineExonDNA AlterationAmino Acid AlterationAllele Frequencyaa3, 5, 6, 8, 13, 15 17, 23, 25, 28, 33, 36,124 G→CLys 8 LysNDbb38, 42, 43, 44, 52, 5938113 G→TAla 5 Ser3%17, 593638 G→AGly 213 Glu9%aPercentage shown represents allele frequency in a general population.bND, not determined.To determine if the divergent sequences represented mutations of the BCL10 gene or polymorphisms, a panel of 50 normal genomic DNA samples from the general population was screened (data not shown). The nucleotide alteration in exon 1 seen in 18 MMs (Table 1Table 1) was assumed to be a polymorphism, because it was observed in numerous MMs and a control sample, and it did not predict an amino acid change. Thus, this variant was not studied further. The nucleotide change in exon 1 (nucleotide 13) of Meso 38 destroys an AciI restriction enzyme site. Restriction enzyme analysis revealed that three of 50 samples from the general population do not possess this AciI site, which corresponds to an allele frequency of 3%. The nucleotide alteration in exon 3 (nucleotide 638) of Meso 17 and Meso 59 does not occur at a restriction enzyme site. Therefore, we performed SSCP analysis on these two MMs and 50 normal DNA samples. Nine of 50 DNA samples from normal individuals (allele frequency, 9%) displayed banding patterns identical to those observed in Meso 17 and Meso 59 (Table 1Table 1). Thus, we conclude that all of the nucleotide differences detected in our MM cell lines and tumors represent polymorphisms in the general population. We did not observe any of the nucleotide alterations reported by Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999. Therefore, it would be important to determine if any of their alterations are polymorphisms.Finally, we performed sequence analysis on DNA from PBL samples matched to the three cell lines (Mesos 17, 38, and 59) that exhibited nucleotide changes predicting amino acid substitutions. The identical nucleotide sequence was observed in DNA from both tumor and normal tissues from the same individual, indicating that these base changes are representative of the constitutional (inherited) genome in each case. In conclusion, BCL10 is not mutated or abnormally expressed in MM, and this gene can be ruled out as a candidate for the 1p22 TSG in this malignancy.‡To whom correspondence should be addressed (e-mail: jr_testa@fccc.edu).

VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5′-aza-2′-deoxycytidine (5′-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients.

Loss of heterozygosity analysis defines a 3-cM region of 15q commonly deleted in human malignant mesothelioma

Previous comparative genomic hybridization and allelic loss analyses demonstrated frequent deletions from 15q11.1–15 in malignant mesothelioma. Recurrent losses of 15q11–22 have also been reported in several other tumor types such as breast and colorectal cancers. To more precisely map the commonly deleted region, we have performed a high density loss of heterozygosity analysis of 46 malignant mesotheliomas, using 26 polymorphic microsatellite markers spanning the entire long arm of chromsome 15. Allelic loss from 15q was observed in 22 of 46 (48%) cases. These analyses have defined a minimally deleted region of ∼3-cM, which was confirmed to reside at 15q15 by fluorescence in situ hybridization analysis with yeast artificial chromosome probes. No tumor suppressor genes have been reported to map to this site. The minimally deleted region identified in this investigation overlaps those observed in other kinds of cancer, and is the smallest site of recurrent 15q loss identified to date in human tumors. The identification of this commonly deleted site implicates a putative tumor suppressor gene(s) at 15q15 involved in diverse forms of human neoplasia.

Molecular cloning, expression and chromosomal localization of a human gene encoding a 33 kDa putative metallopeptidase (PRSM1)

The zincins are a superfamily of structurally-related Zn(2+)-binding metallopeptidases which play a major role in a wide range of biological processes including pattern formation, growth factor activation and extracellular matrix synthesis and degradation. In this paper we report the identification and complete primary structure of a novel 33 kDa protein which contains the zinc-binding HEXXH motif found in the zincin superfamily. We have named this novel protein PRSM1 (PRoteaSe, Metallo, number 1). The gene was identified by the immunoscreening of a human placental cDNA library using polyclonal antibodies raised to the 70 kDa human matrix metalloendopeptidase, type III procollagen N-proteinase [Halila, R. and Peltonen, L. (1986) Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum. Biochem. J. 239, 47-52]. The protein is found in placenta and cultured osteosarcoma cells. PRSM1 could share sequence homology with the type III procollagen N-proteinase. The prsm1 gene is represented once in the human genome and is localized on chromosome 16 (q24.3).

Cancer-targeting siRNA delivery from porous silicon nanoparticles

AIMS Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. METHODS 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. RESULTS Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). CONCLUSION siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

A refined physical map of the long arm of human chromosome 16

Mapping of 33 anonymous DNA probes and 12 genes to the long arm of chromosome 16 was achieved by the use of 14 mouse/human hybrid cell lines and the fragile site FRA16B. Two of the hybrid cell lines contained overlapping interstitial deletions in bands q21 and q22.1. The localization of the 12 genes has been refined. The breakpoints present in the hybrids, in conjunction with the fragile site, can potentially divide the long arm of chromosome 16 into 16 regions. However, this was reduced to 14 regions because in two instances there were no probes or genes that mapped between pairs of breakpoints.