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George Klein

Publications and source records attributed to George Klein.

At least 37 records · Page 2Linked to original sources
PubMed2005

Surveillance against tumors--is it mainly immunological?

The concept of tumor surveillance was first formulated by immunologists Mcfarlane Burnet and Lewis Thomas, respectively. They assumed that the immune system would recognize precancerous and cancer cells as non-self and reject them. This concept is only valid for virally transformed cells, however. In humans, EBV, HHV-8, and the papilloma viruses are relevant viral agents in this context. Tumors arising without a contribution by these viruses are regarded by the immune system as "self", with possible rare exceptions like melanoma. Immunological attempts to influence them therefore implies the breaking of tolerance, a much more difficult proposition. Multicellular organisms have powerful surveillance mechanisms of a non-immunological nature against potential neoplastic cells that threaten to disrupt the organism. Four distinct categories can be recognized: (1) DNA repair. Deficiency of repair enzymes may lead to multiple tumors and/or to multicancer syndromes. (2) Epigenetic mechanisms are currently emerging as being capable of modulating the incidence of certain tumors. e.g., by determining the stringency of imprinting and by influencing chromatin structure. (3) Intracellular surveillance. DNA damage, illegitimate activation of oncogenes, and other pathological changes may activate one or several apoptotic pathways. (4) Intercellular surveillance. The tissue microenvironment influences the probability of disseminated tumor cell growth. Moreover, appropriate differentiation inducing signals may revert the tumor cell phenotype.

Antigens, Viral↗
PubMed2005

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2.

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.

Bacterial Outer Membrane Proteins↗
PubMed2005

Worldwide survey on the methods, efficacy, and safety of catheter ablation for human atrial fibrillation.

BACKGROUND: The purpose of this study was to conduct a worldwide survey investigating the methods, efficacy, and safety of catheter ablation (CA) of atrial fibrillation (AF). METHODS AND RESULTS: A detailed questionnaire was sent to 777 centers worldwide. Data relevant to the study purpose were collected from 181 centers, of which 100 had ongoing programs on CA of AF between 1995 and 2002. The number of patients undergoing this procedure increased from 18 in 1995 to 5050 in 2002. The median number of procedures per center was 37.5 (range, 1 to 600). Paroxysmal AF, persistent AF, and permanent AF were the indicated arrhythmias in 100.0%, 53.0%, and 20.0% of responding centers, respectively. The most commonly used techniques were right atrial compartmentalization between 1995 and 1997, ablation of the triggering focus in 1998 and 1999, and electrical disconnection of multiple pulmonary veins between 2000 and 2002. Of 8745 patients completing the CA protocol in 90 centers, of whom 2389 (27.3%) required >1 procedure, 4550 (52.0%; range among centers, 14.5% to 76.5%) became asymptomatic without drugs and another 2094 (23.9%; range among centers, 8.8% to 50.3%) became asymptomatic in the presence of formerly ineffective antiarrhythmic drugs over an 11.6+/-7.7-month follow-up period. At least 1 major complication was reported in 524 patients (6.0%). CONCLUSIONS: The findings of this survey provide a picture of the variable and evolving methods, efficacy, and safety of CA for AF as practiced in a large number of centers worldwide and may serve as a guide to clinicians considering therapeutic options in patients suffering from this arrhythmia.

Adolescent↗
PubMed2005

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein.

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of the small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.

B-Lymphocytes↗
PubMed2005

Location of Myc, Igh, and Igk on Robertsonian fusion chromosomes is inconsequential for Myc translocations and plasmacytoma development in mice, but Rb(6.15)-carrying tumors prefer Igk-Myc inversions over translocations.

The location of the Myc and immunoglobulin (Ig) loci on metacentric Robertsonian (Rb) fusion chromosomes may affect the development of mouse plasmacytomas (Pcts) by changing the probability with which chromosomal Myc-Ig translocations occur. To test this hypothesis, we induced Pcts in BALB/c (C) mice that carried Rb(4.12) and/or Rb(6.15) chromosomes. The Rb mice developed Pcts (n = 198) with similar onset and incidence to that in the inbred C mice. Karyotyping of 70 Rb-carrying Pcts demonstrated that in these tumors, just as in their counterparts in inbred C mice, the Igh heavy-chain locus was translocated with Myc more often than was the Igk light-chain locus. Pcts harboring Igh or Igk on normal and Rb chromosomes showed no bias toward either in generating Myc translocations. These findings indicated that the location of Myc, Igh, and Igk on normal or Rb chromosomes is inconsequential for Myc translocation and Pct development. In contrast, in Rb(6.15) mice, in which chromosomal inversions competed with chromosomal translocations for Igk-Myc juxtapositions, the former occurred more frequently than the latter in the resulting Pcts. This suggested that spatial proximity of Igk and Myc on the same chromosome facilitates the rearrangement of these loci. Myc translocation-dependent mouse Pct may provide a good model system for furthering our understanding of the relationship of higher-order genome organization in the interphase nucleus, origin of chromosomal translocations, and development of cancer.

Animals↗
PubMed2005

EBV infection induces expression of the transcription factors ATF-2/c-Jun in B lymphocytes but not in B-CLL cells.

B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.

Activating Transcription Factor 2↗
PubMed2004

LIM domains-containing protein 1 (LIMD1), a tumor suppressor encoded at chromosome 3p21.3, binds pRB and represses E2F-driven transcription.

LIM domains-containing protein 1 (LIMD1) is encoded at chromosome 3p21.3, a region commonly deleted in many solid malignancies. However, the function of LIMD1 is unknown. Here we show that LIMD1 specifically interacts with retinoblastoma protein (pRB), inhibits E2F-mediated transcription, and suppresses the expression of the majority of genes with E2F1-responsive elements. LIMD1 blocks tumor growth in vitro and in vivo and is down-regulated in the majority of human lung cancer samples tested. Our data indicate that LIMD1 is a tumor-suppressor gene, the protein product of which functionally interacts with pRB and the loss of which promotes lung carcinogenesis.

Animals↗
PubMed2004

PSK and Trx80 inhibit B-cell growth in EBV-infected cord blood mononuclear cells through T cells activated by the monocyte products IL-15 and IL-12.

Epstein-Barr virus (EBV)-specific immunologic memory is not transferred from mother to child. In vitro infection of cord blood cells can therefore readily lead to the outgrowth of transformed B lymphocytes. We found that the immunomodulator polysaccharide K (PSK) or the mitogenic cytokine truncated thioredoxin (Trx80) inhibited the EBV-induced B-cell proliferation. Using signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) induction as a sign for T- and natural killer (NK) cell activation, we could follow it without any need for cell separation because neither macrophages nor B lymphocytes express SAP. The results suggest the following scenario: EBV infected and activated B lymphocytes. Upon interacting with these cells, T cells became posed for responding to cytokines produced by monocytes. Both PSK and Trx80, which is a secreted C-terminally truncated thioredoxin, activated the monocytes, which then produced cytokines in the presence of the primed T cells. PSK induced interleukin-15 (IL-15), while Trx80 induced IL-12 production. Both cytokines activated the T cells for function. Phosphatidylinositol 3-(PI 3)-kinase and reactive oxygen species (ROSs) were involved in the PSK-induced activation of monocytes. Restimulation of the cultures with EBV-transformed B cells generated specific cytotoxic activity.

Antibodies, Blocking↗
PubMed2004

Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C.

Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the lung cancer region, as a strong candidate tumor suppressor gene. Here we provide additional evidence of the tumor suppressor function of NPRL2/G21. The gene has highly conserved homologs/orthologs ranging from yeast to humans. The yeast ortholog, NPR2, shows three highly conserved regions with 32 to 36% identity over the whole length. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to the MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. The gene is highly expressed in many tissues. We report inactivating mutations in a variety of tumors and cancer cell lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines showed homozygous deletions in the 3' end of NPRL2 in 2 renal, 3 lung, and 1 cervical (HeLa) cell line. Deletions in the 3' part of NPRL2 could result in improper splicing, leading to the loss of the 1.8 kb functional NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signaling, and activation of apoptotic pathway(s). The yeast NPR2 was shown to be a target of cisplatin, suggesting that the human NPRL2/G21 may play a similar role. At least two homozygous deletions of NPRL2/G21 were detected in 6 tumor biopsies from various locations and with microsatellite instability. This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene.

Carcinoma, Renal Cell↗
PubMed2004

Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene.

RASSF1A, a major member of the RASSF1 gene family, is silenced by promoter methylation at a high frequency in a large number of human solid tumors. Controlled expression of RASSF1A reverts the tumorigenic phenotype of several human cancer cell lines. Here we investigated another main isoform, RASSF1C, and compared it with RASSF1A in the gene inactivation test (GIT), based on a tetracycline regulation system. In the small-cell lung cancer (SCLC) line U2020, only RASSF1A has shown growth inhibitory activity in vitro, while in the prostate cell line LNCaP and renal cell carcinoma (RCC) line KRC/Y both RASSF1A and RASSF1C showed similar (approximately 90%) suppressing activity in vitro. Both RASSF1C and RASSF1A suppressed the tumorigenicity of the KRC/Y RCC cell line in SCID mice. Mutations, deletions and loss of expression of RASSF1A and RASSF1C transgenes were identified in all 15 grown SCID tumors. In contrast, the mutant RASSF1A containing Cys65Arg and Val211Ala had reduced growth suppression activity both in vitro and in vivo and did not show any further changes in four grown SCID tumors. In addition, RASSF1C was shown to induce cell cycle arrest in KRC/Y cells. These results strongly imply that like RASSF1A the RASSF1C gene could serve a tumor suppressor function.

Animals↗
PubMed2004

Modern criteria to establish human cancer etiology.

The Cancer Etiology Branch of the National Cancer Institute hosted a workshop, "Validation of a causal relationship: criteria to establish etiology," to determine whether recent technological advances now make it possible to delineate improved or novel criteria for the rapid establishment for cancer causation. The workshop was held in Washington, D.C., December 11-12, 2003, and participants were among the international leaders in the fields of epidemiology, chemistry, biochemistry, microbiology, virology, environmental and chemical carcinogenesis, immunology, pathology, molecular pathology, genetics, oncology, and surgical oncology. There was a general consensus that the rapid identification of human carcinogens and their removal (when possible) or the establishment of specific preventive and therapeutic measures was the most desirable and effective way to have a rapid and positive impact in the fight against cancer. From a clinical perspective, it may be as important to target initiators, cocarcinogens and promoters, if by removing any one of them tumor growth can be prevented. Future studies should focus on interactions among and between different biological, chemical, and physical agents. Analyses of single agents can at times miss their carcinogenic potential when such agents are carcinogenic only in subgroups of individuals because of their genetic background, diet, exposure to other carcinogens, or microbial infection. Epidemiology, molecular pathology (including chemistry, biochemistry, molecular biology, molecular virology, molecular genetics, epigenetics, genomics, proteomics, and other molecular-based approaches), and animal and tissue culture experiments should all be seen as important integrating evidence in the determination of human carcinogenicity. Concerning the respective roles of epidemiology and molecular pathology, it was noted that epidemiology allows the determination of the overall effect of a given carcinogen in the human population (e.g., hepatitis B virus and hepatocellular carcinoma) but cannot prove causality in the individual tumor patient. Molecular pathology cannot determine the overall impact of a carcinogen in the population but can at times prove causality in the individual tumor patient [such as the detection of high-risk human papillomavirus (HPV) in a cervical carcinoma biopsy]. This is possible when molecular techniques have shown that the agent is required for transformation or malignant growth of human cells (such as antisense HPV strategies showing the requirement for the expression of HPV proteins for tumor cell growth) and when there is supportive experimental animal evidence. Ideally, epidemiology and molecular pathology information together with experimental evidence in animals should be available for the most reliable identification of human carcinogens. All sets of data are not always available, and a rapid identification of human carcinogens is in the best public health interest. Swift validation of a causal relationship when followed by a rapid deployment of preventive and therapeutic approaches should lead to a favorable public health impact (such as hepatitis B virus vaccination to prevent hepatocellular carcinoma).

Carcinogens↗
PubMed2004

Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas.

We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.

Breast Neoplasms↗
PubMed2004

RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies.

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G1/S boundary.

Amino Acid Sequence↗
PubMed2004

Microcell-mediated chromosome transfer provides evidence that polysomy promotes structural instability in tumor cell chromosomes through asynchronous replication and breakage within late-replicating regions.

It was reported earlier that normal chromosome 3 (chr3) transfer into tumor cells of different origin may suppress their ability to grow in SCID mice. Tumorigenicity may be restored by the loss of certain 3p regions. We transferred a normal cell-derived chr3 into cells of a human renal cell carcinoma line and followed the chromosomal changes during in vivo and in vitro growth. In cells cultivated for 6 weeks or more and in the tumors grown in SCID mice, supernumerary chrs3 were always rearranged, accompanied by 3p losses. Unexpectedly, we found that the rearrangements affected not only the transferred exogenous chr3, but also the endogenous chrs3. Other chromosomes that were polysomic in the recipient cells were affected as well, suggesting that polysomy may be associated with structural chromosome instability. The dominant chromosomal aberrations were unbalanced translocations with preferentially pericentromeric breakpoints. The breakpoint distribution on chr3 preferentially affected the pericentromeric 3p11 (8 breaks) and 3p12-13 (5 breaks) regions. The regions 3p14 and 3q26-27 occasionally were involved as well (one break in each case). These four regions were the latest replicating, as shown by BrdU incorporation-based replication banding. Using fluorescence in situ hybridization-based replication timing, we detected asynchronous and incomplete centromere replication in cells with 3 or 4 copies of chr3, but not in cells with 2. We concluded that in tumor cells, asynchronous and incomplete replication of polysomic chromosomal parts is associated with aberrations that have breakpoints within the late-replicating regions. This may explain the increased structural chromosome instability and preferential pericentromeric localization of breakpoints in hyperploid tumors.

Aneuploidy↗
PubMed2004

Rearrangements of the telomeric region of mouse chromosome 11 in Pre-B ABL/MYC cells revealed by mBANDing, spectral karyotyping, and fluorescence in-situ hybridization with a subtelomeric probe.

Previous work has demonstrated that chromosome 11 is trisomic or shows a duplicated region encompassing the E1/E2 band of chromosome 11 in 90% of v-abl/myc-induced plasmacytomas (Wiener et al. 1995). In the present report, we have studied BALB/c PreB lymphocytes that were immortalized by v-abl and stably transfected with a conditional MycER vector (Mai et al. 1999). These cells, termed PreB ABL/MYC, showed changes in the E1/E2 bands of chromosome 11 that are similar to those reported previously for v-abl/myc-induced plasmacytomas. This was shown by the use of chromosome painting, SKY, FISH and mBAND. Our findings suggest that the Pre-B ABL/MYC cells may be used to analyse the genetic changes affecting chromosome 11 that are associated with v-abl/myc-dependent tumorigenesis in mouse B cells.

Animals↗
PubMed2004

Modern criteria to determine the etiology of human carcinogens.

Rapid identification of human carcinogens before their dissemination into society, and exposure of worker and lay populations is an important goal of cancer research. Retroactively, verification of in-place human carcinogens is also required to target their removal, and other preventive and therapeutic strategies. The hierarchy of methods used historically for evaluation of carcinogenic potential is epidemiology > animal bioassays > mechanistic studies, and the focus has been on single agents that are genotoxic. However, mechanistic research has revealed several obligatory steps in carcinogenesis, tumor promotion, and progression that can now be used in screening studies with human cells in vitro and animal bioassays. These approaches should be combined with molecular epidemiology and molecular pathology to identify human carcinogens with more emphasis on evaluating combinations of suspect agents and mechanisms of action of epigenetic carcinogens.

Animals↗