Tumor Suppressor Genes CDKN2A Research Articles

Tracking and Treating Malignant Melanoma Metastases

Tracking and Treating Malignant Melanoma Metastases

Molecular dermatopathology in malignant melanoma.

At present, immunohistochemistry is taken for granted in the establishment of malignant melanoma (MM) diagnosis. In recent years, molecular diagnosis in dermatopathology has benefited from a vast array of advances in the fields of genomics and proteomics. Sensitive techniques are available for detecting specific DNA and RNA sequences by molecular hybridization. This paper intends to update methods of molecular cytogenetics available as diagnostic adjuncts in the field of MM. Cytogenetics has highlighted the pathogenesis of atypical melanocytic neoplasms with emphasis on the activation of the mitogen-activated protein kinase (MAPK) signalling pathway during the initiation step of the neoplasms. 20 to 40% of MM families have mutations in the tumour suppressor gene p16 or CDKN2A. In addition, somatic mutations in p16, p53, BRAF, and cKIT are present in MM. Genome-wide scan analyses on MM indicate positive associations for genes involved in melanocytic naevi, but MM is likely caused by a variety of common low-penetrance genes. Molecular dermatopathology is expanding, and its use in the assessment of melanocytic neoplasms appears to be promising in the fields of research and diagnosis. Molecular dermatopathology will probably make its way to an increased number of diagnostic laboratories. The expected benefit should improve the patient management. This evolution points to a need for evolution in the training requirements and role of dermatopathologists.

Genome‐wide linkage analysis of Swedish families to identify putative susceptibility loci for cutaneous malignant melanoma

Cutaneous malignant melanoma is a clinically and genetically heterogeneous disorder which is caused by an interaction between hereditary and environmental factors. In Sweden, a small portion of the inherited susceptibility is explained by the presence of germline mutations in the tumor suppressor gene CDKN2A. But still, the genetic background of melanoma susceptibility is largely unknown. Here, we conducted a genome-wide linkage scan on melanoma-prone families using high-density single-nucleotide polymorphisms (SNPs) arrays to identify novel melanoma susceptibility genes. We investigated 35 families of Swedish origin without CDKN2A mutations. Nonparametric and parametric multipoint linkage analyses were performed. After removal of SNPs in strong linkage disequilibrium, the strongest evidence of linkage was detected on chromosome 17p11-12 (logarithm (base 10) of odds (LOD) scores of 2.76) using parametric linkage analysis assuming a dominant trait with full penetrance. Analyses were also performed on a subset of families with low age at diagnosis (mean age ≤ 47 years), to obtain a more homogenous subset. This subgroup analysis based on 22 families yielded suggestive evidence of linkage to the chromosomal regions 11p12-p11 and 18q22 (multipoint LOD scores of 2.10 and 2.02, respectively). Also, the 17p region that was detected in the complete family set showed suggestive linkage in this cohort (multipoint LOD scores of 2.01). Our data suggest that these chromosomal regions, 17p12-p11 in particular as it was present in both analyses, may harbor genes involved in the susceptibility of malignant melanoma in the Swedish population.

Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: Influence of cruciferous vegetables in an intervention study

Changes in DNA methylation patterns are a hallmark of tobacco-induced carcinogenesis. We have conducted a randomized 4-week intervention trial to investigate the effects of three dietary regimens to modify DNA methylation patterns in peripheral white blood cells of heavy smokers. A group of 88 smokers were randomly assigned to and distributed among three diets, including (1) normal isocaloric diet (balanced in fruits and vegetables), according to international guidelines; (2) a diet enriched in flavonoids and isothiocyanates (particularly cruciferous vegetables); (3) a regimen consisting of diet 1 supplemented with flavonoids (green tea and soy products). Methylation patterns were analyzed by pyrosequencing in LINE1 (Long Interspersed DNA Elements), RASSF1A, ARF and CDKN2a (tumor suppressor genes), MLH1 (mismatch DNA repair) and MTHFR (folate metabolism). Three distinct patterns of methylation were observed. In LINE1, methylation showed a small but reproducible increase with all three regimens. MTHFR was constitutively methylated with no significant modulation by diets. The four other loci showed low basal levels of methylation with no substantial change after intervention. These data suggest that the isocaloric diet may stabilize global epigenetic (LINE1 DNA methylation) patterns in peripheral white blood cells but does not provide evidence for methylation changes in specific genes associated with this short-term dietary intervention.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, Helicobacter pylori infection of the stomach has been correlated with epigenetic changes in the host genome. To identify epigenetic changes during Escherichia coli induced urinary tract infection (UTI), we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC), resulting in intracellular bacteria colonies. Cells inoculated with FimH-negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. UPEC infection significantly induced de novo methyltransferase (DNMT) activity (12.5-fold P=0.002 UPEC vs non-inoculated and 250-fold P=0.001 UPEC vs N-UPEC inoculated cells) and Dnmt1 RNA expression (6-fold P=0.04 UPEC vs non-inoculated cells) compared with controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT-1197) in response to UPEC infection as demonstrated by confocal analysis. Real-time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC-induced downregulation of the tumor suppressor gene CDKN2A (3.3-fold P=0.007 UPEC vs non-inoculated and 3.3-fold P=0.001 UPEC vs N-UPEC) and the DNA repair gene MGMT (9-fold P=0.03 UPEC vs non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8-fold P=0.04 UPEC vs N-UPEC and UPEC vs non-inoculated). Methylation of MGMT was not affected. UPEC-induced methylation of CDKN2A exon 1 may increase bladder cancer and presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence.

1369 UROPATHOGENIC E. COLI INFECTION PROVOKES EPIGENETIC DOWNREGULATION OF CDKN2A (P16INK4A) IN UROEPITHELIAL CELLS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder1 Apr 20111369 UROPATHOGENIC E. COLI INFECTION PROVOKES EPIGENETIC DOWNREGULATION OF CDKN2A (P16INK4A) IN UROEPITHELIAL CELLS Cornelia Tolg, Akshita Kapila, Rene Cortese, Karen Aitken, Arturas Petronis, and Darius Bagli Cornelia TolgCornelia Tolg Toronto, Canada More articles by this author , Akshita KapilaAkshita Kapila Toronto, Canada More articles by this author , Rene CorteseRene Cortese Toronto, Canada More articles by this author , Karen AitkenKaren Aitken Toronto, Canada More articles by this author , Arturas PetronisArturas Petronis Toronto, Canada More articles by this author , and Darius BagliDarius Bagli Toronto, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1193AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, H. pylori infection of the stomach has been correlated with epigenetic changes in the host genome prompting us to investigate whether E. coli induced urinary tract infection (UTI) leads to changes in the host epigenome. Since changes in host cell DNA methylation affect gene expression and persist over several cell generations, DNA methylation may correlate with UTI recurrence and act as biomarker allowing selection of patients that will benefit from antibiotic prophylaxis. METHODS To identify epigenetic changes, we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC) resulting in intracellular bacteria colonies without inducing host cell apoptosis. Cells inoculated with FimH negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. IF, Q-PCR and pyrosequencing were used to analyze expression and methylation of candidate genes. RESULTS UPEC infection significantly induced DNMT activity (12.5 fold p=0.002 UPEC vs. non-inoculated and 250 fold p=0.001 UPEC vs. N-UPEC inoculated cells) and Dnmt1 RNA expression (6 fold p=0.04 UPEC vs. non-inoculated cells) compared to controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT1197) in response to UPEC infection as demonstrated by confocal analysis. Real time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC induced downregulation of the tumour suppressor gene CDKN2A (3.3 fold p=0.007 UPEC vs. non-inoculated and 3.3 fold p=0.001 UPEC vs. N-UPEC) and the DNA repair gene MGMT (9 fold p=0.03 UPEC vs. non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8 fold p=0.04 UPEC vs N-UPEC and UPEC vs. non-inoculated). Methylation of MGMT was not affected. CONCLUSIONS UPEC induced methylation of host cell genes such as CDKN2A exon 1 may presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e546 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cornelia Tolg Toronto, Canada More articles by this author Akshita Kapila Toronto, Canada More articles by this author Rene Cortese Toronto, Canada More articles by this author Karen Aitken Toronto, Canada More articles by this author Arturas Petronis Toronto, Canada More articles by this author Darius Bagli Toronto, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Downregulation of methylthioadenosin phosphorylase by homozygous deletion in gastric carcinoma

The methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 telomeric to the CDKN2A tumor suppressor gene. Loss of MTAP gene is frequently associated with CDKN2A homozygous deletion. Although the homozygous deletion of MTAP has been reported in various human cancers, its function in gastric carcinogenesis is unknown. Here, we determined the status of the MTAP gene by using a combination of array-based comparative genomic hybridization and oligonucleotide microarray. It was found that MTAP was deleted and downregulated in 2 of 10 gastric cancer cell lines. Of the 494 primary gastric carcinomas examined, MTAP expression at the protein level was reduced in 59 (11.9%). Furthermore, a lack of MTAP expression was found to be associated with poor survival (P = 0.038). The genomic loss of MTAP and CDKN2A in gastric carcinomas was investigated by quantitative real-time PCR. Among 20 gastric carcinomas, two cases showed deletion of both MTAP and CDKN2A, and three samples showed homozygous deletion of MTAP, but not of CDKN2A. An analysis of gastric carcinomas revealed that reduced MTAP expression correlated significantly with a genomic deletion. Furthermore, functional assays by transfecting the siRNA or the expressional cDNA into gastric cancer cell lines demonstrated that MTAP regulates cell growth and invasion. The present study suggests that MTAP plays an important role in the regulation of gastric carcinogenesis and, in particular, that MTAP loss is implicated in some way with tumor growth via the modulation of cellular properties, which, in turn, suggests that MTAP has therapeutic applications.

High resolution array comparative genomic hybridization identifies copy number alterations in diffuse large B-cell lymphoma that predict response to immuno-chemotherapy

Despite recent attempts at sub-categorization, including gene expression profiling into prognostically different groups of "germinal center B-cell type" and "activated B-cell type," diffuse large B-cell lymphoma (DLBCL) remains a biologically heterogenous tumor with no clear prognostic biomarkers to guide therapy. Whole genome, high resolution array comparative genomic hybridization (aCGH) was performed on four cases of chemoresistant DLBCL and four cases of chemo-responsive DLBCL to identify genetic differences that may correlate with response to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy. Array CGH analysis identified seven DNA copy number alteration (CNA) regions exclusive to the chemoresistant group, consisting of amplifications at 1p36.13, 1q42.3, 3p21.31, 7q11.23, and 16p13.3, as well as loss at 9p21.3 and 14p21.31. Copy number loss of the tumor suppressor genes CDKN2A (p16, p14) and CDKN2B (p15) at 9p21.3 was validated by fluorescence in situ hybridization and immunohistochemistry as independent techniques. In the chemo-sensitive group, 12 CNAs were detected consisting of segment gains on 1p36.11, 1p36.22, 2q11.2, 8q24.3, 12p13.33, and 22q13.2, as well as segment loss on 6p21.32. RUNX3, a tumor suppressor gene located on 1p36.11 and MTHFR, which encodes for the enzyme methylenetetrahydrofolate reductase, located on 1p36.22, are the only known genes in this group associated with lymphoma. Whole genome aCGH analysis has detected copy number alterations exclusive to either chemoresistant or chemoresponsive DLBCL that may represent consistent clonal changes predictive for prognosis and outcome of chemotherapy.

Pancreatic Ductal Adenocarcinoma and Transcription Factors: Role of c-Myc

Deregulated expression/activation of transcription factors is a key event in the establishment and progression of human cancer. Furthermore, most oncogenic signaling pathways converge on sets of transcription factors that ultimately control gene expression patterns resulting in cancer development, progression, and metastasis. Ductal pancreatic adenocarcinoma (PDA) is the main type of pancreatic cancer and the fourth leading cause of cancer mortality in the Western world. The early stage of the disease is characterized by pancreatic intraepithelial neoplasia lesions bearing mutations in the K-RAS proto-oncogene, which progress to malignant PDA by accumulating additional mutations in the tumor suppressor gene CDKN2A (p16) and in SMAD4 and TP53 transcription factors. The involvement of other signaling pathways in PDA development and progression is an active area of research which may help to clarify the critical steps of this devastating disease. In this regard, several in vitro and in vivo data have demonstrated the contribution of the transcription factor c-Myc to pancreatic carcinogenesis although the molecular mechanisms are poorly understood. c-Myc is a proto-oncogene which has a pivotal function in growth control, differentiation and apoptosis and is known to act as a downstream transcriptional effector of many signaling pathways involved in these processes. It is regulated at multiple levels and its abnormal expression contributes to the genesis of many human tumors. This review focuses on the role of c-Myc in pancreatic embryonic development and homeostasis as well as its involvement on pancreatic tumorigenesis. Evidences showing that c-Myc function is highly dose and cell context dependent, together with its recently demonstrated ability to reprogram somatic cells towards a pluripotent stem cell-like state, indicate that the role of c-Myc in pancreas pathophysiology might have been previously underscored.

Isolation and characterization of a population of stem-like progenitor cells from an atypical meningioma

The majority of meningiomas are benign tumors associated with favorable outcomes; however, the less common aggressive variants with unfavorable outcomes often recur and may be due to subpopulations of less-differentiated cells residing within the tumor. These subpopulations of tumor cells have tumor-initiating properties and may be isolated from heterogeneous tumors when sorted or cultured in defined medium. We report the isolation and characterization of a population of tumor-initiating cells derived from an atypical meningioma. We identify a tumor-initiating population from an atypical meningioma, termed meningioma-initiating cells (MICs). These MICs self-renew, differentiate, and can recapitulate the histological characteristics of the parental tumor when transplanted at 1000 cells into the flank regions of athymic nude mice. Immunohistochemistry reveals stem-like protein expression patterns similar to neural stem and progenitor cells (NSPCs) while genomic profiling verified the isolation of cancer cells (with defined meningioma chromosomal aberrations) from the bulk tumor. Microarray and pathway analysis identifies biochemical processes and gene networks related to aberrant cell cycle progression, particularly the loss of heterozygosity of tumor suppressor genes CDKN2A (p16INK4A), p14ARF, and CDKN2B (p15INK4B). Flow cytometric analysis revealed the expression of CD44 and activated leukocyte adhesion molecule (ALCAM/CD166); these may prove to be markers able to identify this cell type. The isolation and identification of a tumor-initiating cell population capable of forming meningiomas demonstrates a useful model for understanding meningioma development. This meningioma model may be used to study the cell hierarchy of meningioma tumorogenesis and provide increased understanding of malignant progression.

Chromosomal aberrations in iris melanomas

BackgroundUveal melanomas can develop in the choroid, ciliary body and iris. In choroidal and ciliary body melanomas, specific chromosomal changes correlate with metastatic disease. Iris melanomas have a better prognosis...

Analysis of cytogenetic aberrations in sporadic vestibular schwannoma by comparative genomic hybridization

Vestibular schwannomas (VS) are benign tumors of the nervous system that are usually sporadic but also occur in the inherited disorder neurofibromatosis type 2 (NF2). The NF2 gene is a tumor suppressor gene located on chromosome 22. Loss of the NF2 protein product, Merlin, is universal in both sporadic and NF2-related schwannomas and the loss or mutation of the gene is the only established causative event underlying schwannoma formation. Comparative genomic hybridization (CGH) was used to screen 20 sporadic VS to identify additional chromosomal regions that may harbor genes involved in VS-tumorigenesis. The most common change were losses on chromosome 22q. Additionally, losses were observed on chromosome 9p indicating a possible participation of the CDKN2A tumor suppressor gene in the genesis of VS. Gains were observed on 17q, 19p and 19q, which have been reported before in malignant peripheral nerve sheath tumors that are associated with neurofibromatosis type 1. Importantly, high level amplifications have been observed on 16p and 16q as well as on 9q, suggesting the possible involvement of several oncogenes in the tumorigenesis of VS. Our data suggest the involvement of various oncogenes and tumor suppressor genes might play a role in the genesis of the vestibular schwannomas apart from the inactivation of the NF2 gene.

Metastatic Melanoma to the Mandible: Case Report and Review of the Literature

Melanoma is the most serious form of skin cancer. It is a neoplasm of dysplastic pigment-producing melanocytes, and its development is related to the amount of ultraviolet B rays in sunlight that a person is exposed to over his or her lifetime. 1 Fears T.R. Bird C.C. Guerry D. et al. Mean UVB flux and time outdoors predict melanoma risk. Cancer Res. 2002; 62: 3992 PubMed Google Scholar At the molecular level, mutations in the tumor suppressor gene CDKN2A and in the oncogenes N-ras and H-ras seem to play significant roles in its development. 2 Ortonne J.P. Photobiology and genetics of malignant melanoma. Br J Dermatol. 2002; 146: 11 Crossref PubMed Scopus (35) Google Scholar Unlike some other forms of skin cancer, such as basal cell or squamous cell carcinoma, melanomas are highly aggressive and unpredictable, and they can metastasize to almost anywhere in the body.

Pathological classification and molecular genetics of meningiomas

Meningiomas are extremely common adult brain tumors originating from meningeal coverings of the brain and spinal cord. While most are slowly growing Word Health organization (WHO) grade I tumors, rare variants (clear cell, chordoid, papillary, and rhabdoid), as well as brain invasive (WHO grade II), atypical (WHO grade II), and anaplastic (WHO grade III) meningiomas are considerably more aggressive. This review summarizes the histopathological and genetic features of meningiomas, including differential diagnosis, pitfalls, and grading challenges. Early stages of meningioma tumorigenesis are closely linked to inactivation of one or more members of the 4.1 superfamily, including the neurofibromatosis type 2 (NF2) and 4.1B (DAL-1) genes, which interact with the 14-3-3 protein family. Other chromosome 22q genes implicated include BAM22, BCR (breakpoint cluster region), and TIMP-1, the last of which is implicated in higher-grade meningiomas. Atypical meningiomas also commonly show chromosomal losses of 1p, 6q, 10, 14q, and 18q, as well as multiple chromosomal gains. While most relevant genes remain unknown, two chromosome 14q candidates (MEG3 and NDRG2) have recently been identified. In addition to alterations of CDKN2A, p14(ARF), and CDKN2B tumor suppressor genes on 9p21, a contribution of the wingless (wnt) pathway with alterations of the E-cadherin and beta-catenin proteins, as well as alterations of the hedgehog signaling pathway have been implicated in anaplastic meningiomas. The integration of histopathological appearance, complex genetic/genomic data, and outcome will likely result in the identification of clinically distinct meningioma subgroups, which in turn can facilitate the development of targeted therapeutic strategies.

Of patches, blotches, speckles and stripes

From flags to fashion, people love patterns of colors and shapes and use them to convey meaning. Nature is also replete with colorful patterns and employs them to send signals, but how are such patterns generated without our doing? By information or self-organization? John Maynard Smith pointedly asks in his book entitled ‘Shaping Life’. A good question, as the debate has not yet been entirely settled. Proponents of self-organization believe that most if not all repetitive coat patterns can be explained by mathematical models originally based on the reaction-diffusion mechanism proposed by Turing in 1952. Suppose a factor exists in the skin that causes melanocytes to go black, induces an inhibitor that causes them to go white, and is not precisely at equal concentrations at every point on a body surface. Suppose also that the inhibitor diffuses faster than the activator and needs to act before the activator steps in, and – bingo!– there will be black spots surrounded by white areas. One need only tinker with the parameters of an otherwise common algorithm to generate virtually every pattern in nature on a computer screen. Conceptually, then, genes would set the parameters (body size, concentrations, diffusion rates, etc.) and patterns would inevitably and logically emerge. But then there are those who hold that this view underplays the importance of genetic programming, which, after all, underlies all adaptive evolution. They would argue that the pigment cell is not just pushed around by extrinsic factors but is actively engaged in the communication with the local environment and uses its own color genes to draw patterns. As is often the case in such debates, the truth may lie somewhere in between. There is evidence suggesting that patterning genes conceivably influencing reaction-diffusion processes in the integument may have appeared early in evolution and been preserved, and that pigmentation genes interpreting these pattern genes may have evolved later and independently on multiple occasions. This and other questions concerning pigment patterns are discussed in this issue in the commentary by Kaelin and Barsh on the genetics of pigmentation in the cat, by Walker and Gunn in a review on pigment type switching, and by myself in a News and Views piece on sex-specific coloration in fish. Hellström and colleagues from Leif Anderson’s group in Uppsala, Sweden, investigate the genetics of sex-linked barring in chickens and find, intriguingly, that the cell cycle and tumor suppressor gene CDKN2A is involved. Harris and colleagues from the Pavan laboratory at NIH in the US review the various roles SOX proteins play in the development of pigmentation and its regulation in the adult, and they also discuss the involvement of these transcription factors in malignant transformation. Alas, this brings us to pathology, the flip side of all the joy we get from the beauty in animal coloration. In fact, PCMR devotes a substantial number of pages to pathology, for the analysis and treatment of hypo- and hyperpigmentations or pigment cell-associated malignancies. There are several papers in this issue that deal with one or the other factor that is dysregulated in melanoma. Ryan and colleagues, for instance, use comparative genome hybridization to show that the gene encoding Topoisomerase-1 is amplified in aggressive melanoma, giving a poor prognosis. Wang and colleagues show that poor melanoma prognosis is also predicted by aberrant expression of alpha-1 antichymotrypsin. Chen and colleagues show increased expression of Cullin1, which is part of a large ubiquitin E3 ligase complex, in early stage melanoma. VanBrocklin and colleagues use a promising system in mice that makes it possible to evaluate the role of individual genes in melanoma formation alone and in combination. Needless to say, there are many more interesting papers in this issue that should be highlighted, and I encourage you, the reader, to leaf through the pages and mark (and cite!) all the compelling studies that pique your interest.

Id1 overexpression is independent of repression and epigenetic silencing of tumor suppressor genes in melanoma

The full molecular consequences of oncogene activation during tumorigenesis are not well understood, but several studies have recently linked oncogene activation to epigenetic silencing of specific genes 1, 2. Transcriptional repressor Id1 is overexpressed in many malignancies including melanoma, and Id1 targets include tumor suppressor genes TSP1, CDKN2A (p16) and CDKN1A (p21), which are frequently epigenetically silenced in cancer. We confirmed that both TSP1 and CDKN2A have abnormal promoter region DNA methylation in primary melanoma, but the mechanism by which this silencing occurs remains unknown. Here we explore the effects of stable lentiviral Id1 overexpression on the expression of these Id1 target genes in human melanoma cell lines. Overexpressed Id1 was functional and bound transcriptional activator E2A, but did not sequester E2A from gene promoters and repress gene expression. Therefore, these Id1 target genes were resistant to Id1-mediated gene silencing. Our results suggest that Id1 activation may need to occur at discrete stages in cooperation with additional gene dysregulation to repress and induce epigenetic silencing of tumor suppressor genes during melanoma progression.

Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549

BackgroundThe tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.MethodsThe inhibitory effects of p16INK4a, p14ARF and p12 were studied in the human lung cancer cell line A549 which lacks the CDKN2A locus. The eukaryotic expression plasmids of the three transcriptional variants were constructed and stably transfected into the cells. RNA and protein expression by the plasmids was confirmed using RT-PCR and fluorescence immunocytochemistry, respectively. Cell growth inhibition and cell-cycle redistribution after transfection were investigated based on growth curve and flow cytometry analyses. An exogenous His-tag fusion p16INK4a protein was obtained and purified by affinity chromatography. Cell growth inhibition and cell cycle arrest induced by the expression of p16INK4a protein were measured in A549 cells transduced with the exogenous protein.ResultsWhile all three variants suppressed cell growth, p16INK4a had the strongest effect. Marked G1-phase accumulation and S-phase inhibition were induced by p16INK4a and p14ARF but not by p12. Exogenous p16INK4a protein was successfully expressed and purified and transduction of the fusion protein into A549 cells inhibited cell growth by G1→S arrest.ConclusionsAmong the three transcript variants, p16INK4a has a greater inhibitory effect than p14ARF and p12; exogenous p16INK4a protein should be further investigated for use in cancer therapy as a protein agent.

Two Loci Controlling Susceptibility to Radiation-Induced Lymphomagenesis on Mouse Chromosome 4:Cdkn2a, a Candidate for One Locus, and a Novel Locus Distinct fromCdkn2a

BALB/c mice are sensitive to radiation-induced lymphomagenesis, while STS mice are resistant. Using 219 [(BALB/c x STS)F(1) x BALB/c] (N2C) and 197 [(BALB/c x STS)F(1) x STS] (N2S) animals, we performed a genome-wide search for loci controlling susceptibility to lymphomagenesis induced by radiation. Association of markers with the survival of animals was analyzed by the log rank test. For N2C mice, a significant correlation was detected, with four markers on the proximal to mid portion of chromosome 4: D4Mit302 and D4Mit255, P = 0.0075; D4Mit17, P = 0.034; and D4Mit86, P = 0.048. On the other hand, no significant linkage was detected in N2S mice. We analyzed BALB/c mice congenic for the STS allele in different regions of chromosome 4 and identified a locus with a conspicuous effect on survival located within a 7-Mb region between D4Mit302 and D4Mit144, where BALB/c mice harbor hypomorphic variant alleles of the tumor suppressor gene Cdkn2a, which encodes the cyclin-dependent kinase inhibitor protein p16INK4a. Using pooled F(2) intercrosses between the BALB/c and congenic lines carrying the STS allele near D4Mit17, but not in the range from D4Mit302 to D4Mit144, we assigned the second locus to an 11.4-Mb region in the vicinity of D4Mit17. Although Cdkn2a is a likely candidate for the locus controlling susceptibility to lymphomagenesis on chromosome 4, a novel tumor susceptibility gene different from Cdkn2a exists near the primary locus.

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

BackgroundTesticular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs.Materials and methods.Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method.ResultsLOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component.ConclusionsThe higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior.