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.

Selective suppression of lymphomas by functional loss of Hsf1 in a p53-deficient mouse model for spontaneous tumors.

A hallmark in the pathogenesis of cancer is the increased expression of heat shock proteins (Hsps) and other molecular chaperones observed in many tumor types, which is considered to be an adaptive response to enhance tumor cell survival. Heat shock transcription factor 1 (Hsf1) is a major transactivator of Hsp induction and has been proposed to affect tumor initiation and progression, regulating expression of Hsps and other molecular targets. In this report, we provide direct in vivo evidence that Hsf1 plays a critical role in the evolution of spontaneous tumors arising in p53(-/-) mice. Thus, loss of Hsf1 function did not prolong tumor-free survival, but surprisingly altered the spectrum of tumors that arose in p53(-/-) mice. Tumor development is rapid in p53(-/-) mice, which predominantly (about 70%) succumb to lymphomas. In contrast, hsf1(-/-)p53(-/-) mice rarely develop lymphomas (<8%), but succumb to other tumor types including testicular carcinomas and soft tissue sarcomas. Our findings suggest that an increase in p53-independent apoptotic cell death in association with altered cytokine signaling and suppressed production of inflammatory factors in hsf1(-/-) mice may contribute to selective lymphoma suppression. In conclusion, the data presented here link the loss of Hsf1-dependent function to decreased susceptibility to spontaneous lymphomagenesis, which may have implications for cancer prevention and therapy.

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli.

Previous work had identified a corepressor, NAB1, which represses transcriptional activation mediated by NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcription factors are encoded by immediate-early genes and have been implicated in a wide variety of proliferative and differentiative processes. We have isolated and characterized another corepressor, NAB2, which is highly related to NAB1 within two discrete domains. The first conserved domain of NAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of both NGFI-A and Krox20, and its expression is regulated by some of the same stimuli that induce NGFI-A expression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12 cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that is rearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of the Caenorhabditis elegans genome has identified a gene that bears high homology to both NAB1 and NAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role.

claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing.

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.

Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain.

Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.

Deletion and translocation involving chromosome 3 (p14) in two tumorigenic Kaposi's sarcoma cell lines.

BACKGROUND: Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have shown to have normal diploid characteristics are hyperplastic, and depends on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. PURPOSE: We investigated whether the chromosomal changes that occurred in these KS cell lines were random contribute to the pathogenesis of KS. METHODS: We used the conventional G-banding technique and fluorescence in sti hybridization to identify structural and numerical chromosomal changes in the KS cell lines. RESULTS: We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibits loss of heterozygosity of loci at region 3p14-ter. CONCLUSION: This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. IMPLICATIONS: Future research should focus on the identification cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process.

Alterations of the FHIT gene in human hepatocellular carcinoma.

FHIT (fragile histidine triad), a candidate tumor suppressor gene, encompasses FRA3B, a region with the highest fragility in the human genome, and is altered in a large number of human cancers, particularly those of epithelial cell origin and associated with known carcinogenic agents. Human hepatocellular carcinoma (HCC), a major cancer worldwide, is closely related to carcinogenic agents such as hepatitis B and C virus infections, dietary aflatoxin, alcohol consumption, and exposure to chemical carcinogens. To assess the extent and the nature of the FHIT gene alterations and their implications in the development of HCC, several cell lines and primary tumors were cytologically and molecularly examined. The FHIT gene is expressed in normal hepatic cells and is not expressed or is abnormally expressed in cultured tumor cells derived from HCC. Down-regulation of the FHIT gene was detected by Northern blot analysis in 9 of 14 cell lines However, neither abnormal FHIT transcripts nor point mutations in DNA sequences of reverse transcription-PCR products (exons 2-9) were identified. Expression of FHIT protein was not detected by immunostaining in 5 of 10 primary tumors. Four cell lines showing mRNA down-regulation did not express FHIT protein as demonstrated by Western blot analysis. Allelic loss of intron 5 of the FHIT gene was detected in 10 of 34 informative samples from primary tumors. Structural alterations of chromosome 3p were identified in 8 of 13 HCC cell lines. Deletions or translocations involving region 3p14.2 were identified by fluorescence in situ hybridization with a YAC850A6 probe spanning the FHIT locus on chromosomes derived from cell lines with an abnormal FHIT gene expression. These combined results indicate that the FHIT gene is a frequent target and may be implicated in a subset of liver cancers.

Positions of chromosome 3p14.2 fragile sites (FRA3B) within the FHIT gene.

The FHIT gene spans approximately 1 Mb of DNA at chromosome band 3p14.2, which includes the familial renal cell carcinoma chromosome translocation breakpoint (between FHIT exons 3 and 4), the most frequently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions in various human cancers, frequently involving FHIT exon 5. The FRA3B has previously been shown to represent more than one specific site, and some specific representatives of FRA3B breaks have been shown to fall in two regions, which we know to be in FHIT introns 4 and intron 5. Because breakage and integration of exogenous DNA in this chromosome region is frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or breaks, we determined by one- and two color fluorescence in situ hybridization, using cosmids covering specific regions of the FHIT gene, that most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 of the FHIT gene, less than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where a human papillomavirus 16 integration site has been localized, and in intron 3, where the t(3;8) break point is located. These results suggest that the cancer-specific deletions, which frequently involve introns 4 and 5, originated through breaks in fragile sites.

Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP.

The isolation of genes involved in cancer development is critical for uncovering the molecular basis of cancer. We report here the isolation of the full-length cDNA and chromosomal localization of a new gene frequently deleted in liver cancer (DLC-1) that was identified by representational difference analysis. Loss of heterozygosity was detected for DLC-1 in 7 of 16 primary hepatocellular carcinomas (HCCs) and in 10 of 11 HCC cell lines. Although mRNA for DLC-1 was expressed in all normal human tissues, it was not expressed in 4 of 14 HCC cell lines. Full-length cDNA for DLC-1 of 3800 bp encodes a protein of 1091 amino acids, has 86% homology with rat p122 RhoGAP gene, and was localized by fluorescence in situ hybridization on chromosome 8 at bands p21.3-22. Deletions on the short arm of chromosome 8 are recurrent in liver, breast, lung, and prostate cancers, suggesting the presence of tumor suppressor genes. DLC-1 may be a tumor suppressor gene in liver cancer as well as in other cancers.

Localization of the human HER4/erbB-4 gene to chromosome 2.

The HER4/erbB-4 gene has been isolated as the fourth member of the human EGFR subfamily of tyrosine kinases and has been reported to encode a receptor for NDF/heregulin. In the present study we determined the chromosomal location of the HER4/erbB-4 gene within the human genome. Using human cDNA probes in fluorescence in situ hybridization (FISH), we mapped the HER4/erbB-4 gene to human chromosome 2q33.3-34. This finding established that also the HER4/erbB-4 gene is located in close vicinity of homeobox and collagen gene loci, as is the case for the related EGFR, erbB-2/neu and erbB-3. Aberrations of this chromosomal region associated with T cell leukemias and lymphomas as well as alveolar rhabdomyosarcomas raise the possibility that HER4/erbB-4 might be activated in these tumour types.

Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13.

The mammaglobin gene encodes a novel secreted protein whose corresponding mRNA is frequently up-regulated in human breast cancer. In non-malignant tissues, expression is also strictly limited to the mammary epithelium. To better understand the mechanisms controlling these patterns of expression, we have isolated the human mammaglobin gene and performed an initial assessment of its promoter activity. Mammaglobin gene architecture is very similar to that of a family of related genes that includes uteroglobin and rat prostatein subunits C1, C2, and C3. However, the mammaglobin gene itself is not well conserved phylogenetically. The human mammaglobin gene is localized by fluorescent in situ hybridization to chromosome 11 band q13, a genomic region frequently amplified in breast neoplasia. The sequence of proximal 1 kb of mammaglobin promoter contains several potential transcriptional control elements and directs high-level expression of a transfected reporter construct in human breast tumor cell lines. However, comparable levels of reporter gene expression are also seen in non-mammary human cell lines. These data suggest that, unlike related gene family members, the striking breast-specific expression and tumor-associated overexpression of mammaglobin is mediated by complex transcriptional control at more distal sequence elements.

Expression of alternative forms of Ras exchange factors GRF and SOS1 in different human tissues and cell lines.

DNA probes and antibodies specific for different coding regions of human SOS1 and GRF genes were used to screen expression of these genes in a variety of adult and fetal human tissues and cell lines. Despite previous reports of the exclusive expression of hGRF RNA in brain, we also observed expression of this gene in various other tissues including lung and pancreas, as well as several tumor cell lines. At least three different hGRF mRNA transcripts were observed depending on the probe used, with the larger transcripts being detected by probes corresponding to the 5' end of the gene while smaller transcripts were detected by probes corresponding to the 3' end. Expression of hSOS1-related transcripts was more ubiquitous and homogeneous than with hGRF, with similar levels of specific transcripts being detected in most tissues and cell fines tested. Three to five different transcripts were detected in human tissues when using probes for the 5' end and middle regions of this gene, whereas only two were detected with probes corresponding to the 3' end. Screening of multiple human tumor cell lines showed ubiquitous expression of three specific transcripts, although the level and ratio of each of these transcripts varied widely among individual cell lines. Consistent with the variety of transcripts detected, several protein forms were also identified in Western immunoblots with antisera raised against specific domains of hSOS1 and human Ras-GRF gene products. Fluorescence in situ chromosomal hybridization suggested that, in both cases, the multiple forms arise from single chromosomal loci. The heterogeneity of hGRF and hSOS1 gene products detected (which appear to retain in most cases a functional catalytic domain), suggests that differentially expressed, alternatively spliced hSOS1 and hGRF forms may contribute to fine regulation of Ras activation in different tissues or at different stages of development.

Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X.

Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

Novel genomic imbalances and chromosome translocations involving c-myc gene in Burkitt's lymphoma.

In this study, CA46 and ST486, two Epstein-Barr (EBV) negative cell lines derived from sporadic BL, were analyzed by multicolor spectral karyotyping, G-banding, fluorescence in situ hybridization with single-copy gene probes, and comparative genomic hybridization (CGH). In addition to reciprocal t(8;14)(q24;q32) translocation involving c-myc and IgH loci, we identified a t(7;8;14)(q11.2;q24;q32) translocation in CA 46 cells and t(8;14;18)(q24;q32;q23) in ST486 cells. Both rearrangements were not previously described in BL and resulted in transposition of myc sequences in a new genomic configuration. Several DNA imbalances mapped by CGH at the same sites in both lines, may reflect recurrent genomic changes that are relevant to pathogenesis of BL. We tested the tumorigenicity of these lines by injecting cells intraperitoneally in SCID mice. In two separate experiments, CA46 cells produced tumors 2 weeks after cell inoculation while ST486 cells induced only one tumor after a long latency period. Partial duplication of the long arm of chromosome 1 involving variable bands but always band 1q23 is the second most common alteration in BL and is known to be associated with aggressive tumors and poor prognosis. Duplication of the bands 1q23-24 commonly observed in EBV-negative lines was identified only in highly tumorigenic CA46 cells suggesting that this region harbor gene(s) associated with tumor cell invasiveness.

A chromosome 4 satellite I DNA isolated from SV40-transformed human cells.

Analysis of cellular DNA insert isolated from a free replicative plasmid rescued from human cells transformed with an SV40 vector plasmid revealed the presence of two arrays of repetitive DNA arranged in tandem. One sequence was homologous to the consensus sequence of the human alpha satellite DNA and the adjoining sequence was a satellite DNA sequence which consisted of repetitive units of 42 base pairs (bp) and was designated HR42. The degree of homology between repetitive units was about 92%. By Southern analysis the HR42 sequence was detected in HHW416, a somatic cell hybrid containing human chromosome 4, but not in HDm-5, the somatic cell hybrid which has human chromosome 14. By fluorescence in situ hybridization this repetitive DNA was assigned uniquely to the centromeric region of human chromosome 4. These results show that HR42 belongs to a subfamily of satellite I DNA specific for human chromosome 4.

Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2).

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.-F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.

Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis.

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.

Profile of genetic alterations and tumorigenicity of human breast cancer cells.

The profile of genetic alterations in four breast carcinoma cell lines, SK-BR-3, BT-474, MDA-MB361 and ZR-75-1 was examined by comparative genomic hybridization, G-band karyotyping, reverse chromosome painting and fluorescence in situ hybridization of single-copy genes. These lines are aneuploid with complex structural rearrangements and have DNA copy-number imbalances involving multiple sites that include amplification of ERBB-2 and MYC proto-oncogenes which are implicated in breast cancer pathogenesis. A novel site of high level amplification was mapped on chromosome 15. All lines were tumorigenic in nude mice, however, the latency and the incidence of tumor formation varied; SK-BR-3 and MDA-MB361 produced tumors in a shorter time and had a higher total number of genomic imbalances compared to BT-474 and ZR-75-1 cells. Tumor cell behavior in vivo was not reflected by the rate of in vitro cell proliferation. Underrepresentation on the long arm of chromosome 18 was the sole alteration that correlated with an increased tumorigenicity. Chromosome 18q is rich in tumor suppressor genes and its loss is prevalent in primary node-positive breast tumors. Cell lines with monoclonal populations preserve the genetic characteristics of the primary tumor and their use may facilitate the detection of specific alterations associated with breast cancer progression.

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.