Integration of Epstein-Barr virus into chromosome 6q15 of Burkitt lymphoma cell line (Raji) induces loss of BACH2 expression

Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is a well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50-60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly guanine/cytosine-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent host sequences. EBV was present at intron 1 of the BACH2 gene located on chromosome 6q15. BACH2 was not expressed in the Raji cell line. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis.     

Identification of Epstein-Barr virus integrated sites in lymphoblastoid cell line (IB4)

IB4 is a lymphoblastoid cell line frequently used for the functional analysis of the latent genes of EBV. Previous study indicated that EBV whole genome is integrated tandemly as the linear viral genome into host genome of IB4, although sites of integration have not been determined. Through cloning of the junctional regions between EBV and host genomes, one of the integration sites was identified on the BamHI-C fragments around oriP sequences and another on the EcoRI-I fragment. Both of the integration sites were located on the clone RP11-119H12 of chromosome 4q25 and separated approximately 6.5 kbp from each other. The integration sites identified were apart from the genes of the host genome, indicating that both host gene and EBV latent genes are not altered by the integration event.     

Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes, and cancer chromosome breakpoints

The integration sites of human papillomavirus (HPV) DNA within the cervical carcinoma cell line C4-I and a primary cervical tumor were mapped by in situ hybridization. Cloned cellular sequences flanking the integrated viral DNA were used as probes. For the cell line, the viral integration site was mapped to chromosome region 8q21-q22.3, while in the primary tumor chromosome band 3p21 was the target for integration. The HPV DNA integration appears to occur in the vicinity of fragile sites, oncogenes, and chromosome breakpoints that are characteristic of hematologic malignancies and solid tumors. The integration of HPV may thus promote chromosome changes in cancer cells.     

Amplicons on chromosome 3 contain oncogenes induced by recurrent exposure to 12-O-tetradecanoylphorbol-13-acetate and sodium n-butyrate and Epstein-Barr virus reactivation in a nasopharyngeal carcinoma cell line

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection and exposure to environmental carcinogens. In this study, an inducible Epstein-Barr virus (EBV) reactivation NPC cell line, NA, was used to investigate the impact of recurrent 12-O-tetradecanoylphorbol-13-acetate-sodium n-butyrate (TPA/SB) treatment and EBV reactivation on chromosomal abnormalities utilizing array-based comparative genomic hybridization (CGH). It was observed that most copy-number aberrations (CNA) were progressively nonrandomly clustered on chromosomes 3, 8, and 9, as the frequency of TPA/SB treatment and EBV reactivation increased. All of the prominent amplicons detected (including 3p14.1, 3p13, 3p12.3, 3p12.2, 3q26.2, 3q26.31, and 3q26.32) were located on chromosome 3, with multiple oncogenes assigned to these sites. The amplification patterns of 3p12.3 and 3q26.2 were validated using fluorescence in situ hybridization (FISH) analysis. Subsequent quantitative real-time polymerase chain reaction detected increasing expression of ROBO1 and SKIL oncogenes in NA cells harboring higher frequency of TPA/SB treatment and EBV reactivation, consistent with copy-number amplification of these loci. These findings demonstrate that a high incidence of TPA/SB induced-EBV reactivation has a profound influence on the carcinogenesis of NPC through altered DNA copy number.     

Human herpesvirus 6 integrates within telomeric regions as evidenced by five different chromosomal sites

Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal integration sites of human herpesvirus 6 (HHV-6) in phytohemagglutinin-stimulated leukocytes and B lymphocytes from Epstein-Barr virus transformed lymphoblastoid cell lines (LCLs). Five different chromosomal integration sites were found in nine individuals. Only one site was identified in each individual, each site was in the vicinity of the telomeric region and was on either the p or q arm of only one of the two chromosome homologues. The sites were 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4, of which three have not been previously identified. For 9q34.3 the site of integration was further mapped using a locus-specific probe for 9q34.3 together with a pan-telomeric probe and both co-localized with the HHV-6 signal. Similarly an arm-specific telomeric probe for 19q co-localized with the HHV-6 signal. It was therefore concluded that the site of integration is actually within the telomere. The number of viral DNA copies/cell was calculated in blood, LCL cells and hair follicles and was one or more in every case for each of the nine individuals. This result was confirmed by FISH where 100% of cells gave an HHV-6 signal. These findings add to previous reports suggesting that integrated HHV-6 DNA is found in every cell in the body and transmitted vertically. Finally, including our data, worldwide seven different chromosomal sites of HHV-6 integration have now been identified. Large epidemiological studies of chromosomal integration are required to identify further telomeric sites, geographical or racial variation and possible clinical consequences.     

Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji)

Epstein-Barr virus (EBV) integration into a Burkitt's lymphoma (BL) cell line (Raji) was investigated, using polymerase chain reaction (PCR), Southern hybridization, genomic library screening and fluorescence in situ hybridization (FISH). BaMHIW fragments of the EBV genome and DNA sequences of the viral latent membrane protein (LMP)1 and LMP2 genes were detected in Raji cells. BaMHI-digested high-molecular weight DNA from Raji cells generated 4 and 10 kb, 23 kb fragments that hybridized to Probe-1 (EBV genome 13232-16189) and Probe-2 (EBV genome 5-3271). Genomic library for Raji cells was constructed. Plaques (1 x 10(5)) were screened with Probe-2, and four positive clones were obtained. Chromosomal integration of EBV DNA was detected in the Raji cell. The viral integration sites included 1p, 1q, 2q, 3p, 3q, 4q, 5q, 6q, 7p, 7q, 9q, 11p, 14q and 15q. Despite this multiplicity of integration sites, integration showed high frequency only at the sites 4q, 2q, 1q and 7q; 64% of the total signals were found in these four chromosomal bands. No viral integration occurred in chromosomes 16-22 or the sex chromosomes (X, Y). This study is the first comprehensive FISH analysis of EBV integration into the chromosomes of the Raji cell line. The findings support the notion that EBV integrates into the Raji cell genome non-randomly.     

Detection of Epstein-Barr virus-infected mucosal lymphocytes in nasal polyps.

Primary nasal lymphomas of T or NK cell origin are known to be associated with Epstein-Barr virus (EBV). However, it is not known whether EBV is normally present in nasal mucosa as distinct to nasopharyngeal tissue. This study investigates the prevalence of EBV infection in 13 cases of nasal polyps. EBV DNA was detected in 2 of 13 (15%) by Southern blot hybridization and in 9 of 13 (69%) by polymerase chain reaction. In situ hybridization for EBV-encoded small nuclear RNAs (EBER) was positive in 11 of 13 (85%) cases; the virus was present in stromal lymphocytes only and not in the epithelial cells. Immunohistochemistry for EBV proteins in 7 cases revealed EBV nuclear antigen (EBNA)-2, latent membrane protein (LMP)-1, and ZEBRA (the switch protein encoded by gene BZLF1) expression in rare isolated stromal lymphocytes in 3 cases. Double immunostaining in 1 case showed that the LMP-1+ cells were B or T cells. Immunohistochemistry for EBV lytic proteins showed very rare viral capsid antigen (VCA)+ and membrane antigen (MA)+ cells in 1 case and very rare diffuse early antigen (EA-D)+ and VCA+ cells in 1 other case. The expression of ZEBRA, EA-D, VCA, and MA suggested a disruption of latency in very rare stromal lymphocytes leading to a productive cycle. Although the incidence of EBV positivity in nasal polyps in our population is high (85%), very low numbers of EBV+ cells are found in each case. Nevertheless, they indicate that nasal mucosa could be one of the sites of EBV persistence through a low level of infection of the resident lymphocytes and thereby provide a possible setting for the emergence of virally associated tumors in this site.     

Cytogenetic study of chronic lymphocytic leukemia in ten Japanese patients with a case of the same chromosome abnormality both in T and B cells

Peripheral blood lymphocytes from ten patients with chronic lymphocytic leukemia (CLL) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Epstein-Barr virus (EBV), and T-cell growth factor were studied cytogenetically. Eight of ten cases were clinically and immunologically B-cell CLL, and two cases were T-cell CLL. Chromosome analysis revealed that one patient, diagnosed as B-cell CLL, had a clonal abnormality in the leukemic cells. The abnormal karyotype was 46,XY,+3,-15,t(10;19)(q22;p13),t(11;14) (q13;q32),13q+,17q-. The frequencies of the abnormal cell stimulated with PHA, PWM, and EBV were 50%, 13%, and 100%, respectively. Finding the same clonal abnormality after stimulation with three different mitogens suggests that the factor responsible for the development of CLL might affect the stem cell common to T-cell and B-cell lymphocytes. In cells with this abnormal karyotype, chromosome 13q+ seems to have a homogeneous staining region. In the present series of B-cell CLL, no trisomy 12 known to be the specific chromosome abnormality was observed.     

Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization

Metaphase chromosomes of three cervical cancer cell lines (HeLa, CasKi, SiHa) were subjected to in situ hybridizations with the DNA of human papillomaviruses (HPV) types 16 and 18, respectively. Previous studies have demonstrated multiple copies of HPV 18 DNA in HeLa and of HPV 16 DNA in CasKi cells, but only 1–2 HPV 16 copies in cells of the SiHa line. The viral DNA persists in an integrated state (Schwarz et al 1985). Analysis of the integration sites revealed at least 11 chromosomal sites of HPV 16 integration in CasKi cells. SiHa cells contain integrated HPV 16 DNA in the region q21–q31 of chromosome No. 13. In HeLa cells integration of HPV 18 occurred in chromosome No. 8, band q24. Thus, no evidence was obtained for the existence of preferential chromosomal regions for HPV integration. The data indirectly support a trans-acting function of HPV-mediated cell transformation.On leave of absence from the Institute of Infectious and Parasitic Diseases, Medical Academy, VI. Zaimov 26, 1504 Sofia, Bulgaria     

Losses of 3p, 11p, and 13q in EJ/ras-transformable simian virus 40-immortalized human uroepithelial cells.

Five independent clones of Simian virus 40 (SV40)-immortalized human uroepithelial cells (CK/SV-HUC) were established after transfection of HUC cultures from the same tissue donor with plasmids encoding SV40 large T and small t antigen genes. Each CK/SV-HUC clone contained a unique SV40 integration site, and all expressed similar levels of SV40 mRNA. All five clones were nontumorigenic, but clones 2, 4, and 5 tumorigenically transformed after transfection at P19 with mutant EJ/ras and also spontaneously after 40 serial passages in vitro. In contrast, CK/SV-HUC clones 1 and 3 did not transform when either approach was used. These differences in transformability among CK/SV-HUC clones could not be predicted based on differences in SV40 gene expression nor on any in vitro growth property tested. In cytogenetic analyses, a transformable clone showed losses of three chromosome arms containing putative cancer suppressor gene regions, including 3p14----pter, 13q, and 11p, whereas the nontransformable clones showed none of these losses. Thus these data indicate that genetic losses on 3p, 11p, and 13q may contribute to tumorigenic transformation of SV40-immortalized human uroepithelial cells.     

U-HO1. A new cell line derived from a primary refractory classical Hodgkin lymphoma

The Hodgkin cell line U-HO1 was established from a malignant pleural effusion of a 23-yr-old male patient during the end stage of refractory nodular sclerosing classical Hodgkin lymphoma (cHL). Since its establishment in 2005, U-HO1 has maintained stable characteristics in vitro and has a doubling time of about 4 days under standard culture conditions. U-HO1 forms typical Reed/Sternberg cells in suspension, is EBV negative, lacks HLA-ABC- but expresses HLA-D- proteins/CD74 and surface exposes CD15 together with CD30 in the absence of CD19 and CD20. Karyotype analysis of U-HO1 revealed a hyperdiploid karyotype with multiple clonal aberrations. Most significant is an elongated chromosome 2, der(2)t(2;10)(q35;q16.1)add(2)(p13). CGH analysis revealed the following imbalances: ish cgh dim(1)(p13p31)(p12q21), enh(2)(p13p23), dim(4)(q31.3qter), enh(6)(q22q27), enh(12), enh(18),enh(20)(q13.1pter). FISH analysis showed about six-fold amplification of REL and BCL-11A, thus, U-HO1 is prototypical for cHL in every aspect tested so far. Compared to other HL cell lines, U-HO1 proved far less genetically aberrant suggesting that U-HO1's imbalances suffice to cause the full-blown phenotype of primary refractory cHL.     

Nature of distamycin A-inducible fragile sites

Five rare distamycin A-inducible fragile sites have been identified on human chromosomes: fra(8)(q24.1), fra(11)(p15.1), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites are located at the junction of Giemsa-positive (G) and negative (R) bands and their expression can be induced by a variety of AT specific DNA ligands. Analysis of family data indicate that the distamycin A-inducible fragile sites segregate as a simple codominant trait with complete penetrance, and probands receive these fragile site genes equally from mothers and fathers. Based on current knowledge of chromosome instability, the nature of distamycin A-inducible fragile sites is discussed. Distamycin A-inducible fragile sites appear to be unique chromosomal regions particularly susceptible to fragility under certain stress conditions. They may also be hot spots for recombination, gene amplification, and integration of foreign genomes.     

Contiguous arrangement of p45 NFE2, HnRNP A1, and HP1 alpha on mouse chromosome 15 and human chromosome 12: evidence for suppression of these genes due to retroviral integration within the Fli-2 locus

Fli-2 is a common site of proviral integration in multistage erythroleukemia cells induced by Friend murine leukemia virus (F-MuLV) or the polycythemia strain of Friend leukemia virus (FV-P). Previously, we reported that integration of Friend virus into the Fli-2 locus in CB3, an erythroleukemia cell line that harbors a homozygous inactivation of the Fli-2 locus, results in the loss of expression of two genes encoding the 45-kDa subunit of the erythroid-specific nuclear factor p45 NFE2 and the splicing factor HnRNP A1. Here, we report the identification of a third gene, Heterochromatin protein 1 (HP1alpha, also known as CBX5), which is located downstream of HnRNP A1, and p45 NFE2. Northern blot analysis revealed that the expression of HP1alpha, along with p45 NFE2 and HnRNP A1, is either undetectable or substantially reduced in CB3 cells, suggesting that HP1alpha expression is also regulated by proviral insertion within the Fli-2 locus in CB3 cells. Because p45 NFE2 was previously mapped to mouse chromosome 15, our results demonstrate that HP1alpha and HnRNP A1 are also located on mouse chromosome 15 and that the p45 NFE2, HnRNP A1, and HP1alpha genes are arranged contiguously. Contiguous arrangement of these three genes was also detected in man; this consequently localizes HP1alpha to human chromosome band 12q13.     

Fine mapping of an apparently targeted latent human herpesvirus type 6 integration site in chromosome band 17p13.3

An unusually high level of latent HHV-6 infection has been documented in the peripheral blood and/or bone marrow cells of a small group of patients with predominantly malignant lymphoid disorders, and in at least one healthy individual. We have shown previously in peripheral blood mononuclear cells (PBMCs) of three patients, two with a history of lymphoma and one with multiple sclerosis, a specific target site for latent integration of the full-length HHV-6 viral genome on the distal short arm of chromosome 17, in band p13.3. Fluorescence in situ hybridization (FISH) procedures were used to map more precisely the location of the viral integration site in one of those patients, relative to two known oncogenes mapped previously, namely CRK, and the more telomeric ABR oncogene. It is shown that the HHV-6 integration site is located at least 1,000 kb telomeric of ABR, and is very likely to map close to or within the telomeric sequences of 17p. This finding is significant given that human telomeric-like repeats flank the terminal ends of the HHV-6 genome. Cytogenetic studies showed evidence of karyotype instability in the peripheral blood cells infected latently.     

Analysis of ovarian cancer cell lines using array-based comparative genomic hybridization

In this study, 23 ovarian cancer cell lines were screened using array-comparative genomic hybridization (aCGH) based on large-insert clones at about 1 Mb density from throughout the genome. The most frequent recurrent changes at the level of the chromosome arm were loss of chromosome 4 or 4q, loss of 18q and gain of 20 or 20q; other recurrent changes included losses of 6q, 8p, 9p, 11p, 15q, 16q, 17p, and 22q, and gain of 7q. Losses of 4q and 18q occurred together more often than expected. Evidence was found for two types of ovarian cancer, one typically near-triploid and characterized by a generally higher frequency of chromosomal changes (especially losses of 4p, 4q, 13q, 15q, 16p, 16q, 18p and 18q), and the other typically near-diploid/tetraploid and with fewer changes overall, but with relatively high frequencies of 9p loss, 9q gain, and 20p gain. Multiple novel changes (amplifications, homozygous deletions, discrete regions of gain or loss, small overlapping regions of change and frequently changed clones) were also detected, each of which might indicate the locations of oncogenes or tumour suppressor loci. For example, at least two regions of amplification on chromosome 11q13, one including cyclin D1 and the other the candidate oncogene PAK1, were found. Amplification on 11q22 near the progesterone receptor gene and a cluster of matrix metalloproteinase loci was also detected. Other potential oncogenes, which mapped to regions found by this study, included cyclin E and PIK3C2G. Candidate tumour suppressor genes in regions of loss included CDKN2C, SMAD4-interacting protein and RASSF2.     

Genetic alterations during the progression of squamous cell carcinomas of the uterine cervix.

Most cervical carcinomas appear to arise from cervical intraepithelial neoplasia (CIN) lesions. In addition to infection with high-risk human papilloma viruses, which is indicative of an increased risk of progression, alterations of oncogenes and tumor suppressor genes play a role. Genetic studies of CIN lesions, primary cervical carcinoma, and metastases may shed light on the relative importance of various genetic alterations involved in the progression of CIN to invasive carcinoma. We examined tumor material from 10 patients with squamous cell carcinoma of the uterine cervix and synchronous CIN lesions and lymph node metastases. The CIN component, invasive carcinoma, and lymph node metastases were analyzed separately for loss of heterozygosity (LOH) on the following loci: VHL (3p21), HLA region (6p22-23), PGL (11q 22-24), E6 associated protein (15q11-13), TP53 (17p13), DCC (18q21.1), and chromosomes 1, 2, 4, 9, 20, and X. Using immunohistochemistry, the expression of the EGF receptor, ERBB2, and TP53 was determined. In CIN lesions, frequent LOH was found at chromosome arms 3p, 6p, and 11q. Primary invasive carcinoma showed additional LOH at chromosome arms 6q, 17p, and 18q. In lymph node metastases, an additional locus on the X chromosome displayed LOH. All carcinomas and synchronous lesions but one showed high expression levels of the EGF receptor. TP53 staining, when present, was found in all synchronous lesions. Focal staining of ERBB2 was found in one CIN lesion, two invasive carcinomas, and four metastases. The molecular alterations accumulated in a fashion that paralleled the progression of the tumors. These results indicate that cervical tumorigenesis occurs in a stepwise fashion, including infection and integration of oncogenic HPV and several specific genetic alterations. Genes Chromosomes Cancer 26:346-354, 1999.