Control of protein function through oxidation and reduction of persulfidated states.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Cell size regulation, a mechanism that controls cellular RNA accumulation: consequences on regulation of the ubiquitous transcription factors Oct1 and NF-Y and the liver-enriched transcription factor DBP.

Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA-equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level.

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.

Image of a sulfur atom.

Two-dimensional signal averaging has been applied to dark field electron micrographs of molecules of 2,3,4,5-tetraacetoxymercurithiophene. Only the mercury atom images are seen in single micrographs. However, in the composite image, resulting from photographic superposition of 64 individual images, the sulfur atom in the molecule is clearly revealed.

A Generic Method and Implementation to Evaluate and Improve Data Quality in Distributed Research Networks.

BACKGROUND: With the increasing personalization of clinical therapies, translational research is evermore dependent on multisite research cooperations to obtain sufficient data and biomaterial. Distributed research networks rely on the availability of high-quality data stored in local databases operated by their member institutions. However, reusing data documented by independent health providers for the purpose of care, rather than research ("secondary use"), reveal a high variability in terms of data formats, as well as poor data quality, across network sites. OBJECTIVES: The aim of this work is the provision of a process for the assessment of data quality with regard to completeness and syntactic accuracy across independently operated data warehouses using common definitions stored in a central (network-wide) metadata repository (MDR). METHODS: For assessment of data quality across multiple sites, we employ a framework of so-called bridgeheads. These are federated data warehouses, which allow the sites to participate in a research network. A central MDR is used to store the definitions of the commonly agreed data elements and their permissible values. RESULTS: We present the design for a generator of quality reports within a bridgehead, allowing the validation of data in the local data warehouse against a research network's central MDR. A standardized quality report can be produced at each network site, providing a means to compare data quality across sites, as well as to channel feedback to the local data source systems, and local documentation personnel. A reference implementation for this concept has been successfully utilized at 10 sites across the German Cancer Consortium. CONCLUSIONS: We have shown that comparable data quality assessment across different partners of a distributed research network is feasible when a central metadata repository is combined with locally installed assessment processes. To achieve this, we designed a quality report and the process for generating such a report. The final step was the implementation in a German research network.

Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line.

Dihydrofolate reductase (DHFR) enzyme is preferentially synthesized in proliferative cells. A mouse muscle cell line resistant to 300 microM methotrexate was developed to investigate the molecular levels at which DHFR is down-regulated during myogenic withdrawal from the cell cycle. H- alpha R300T cells contained 540 copies of the endogenous DHFR gene and overexpressed DHFR mRNA and DHFR protein. Despite DHFR gene amplification, the cells remained diploid. As H- alpha R300T myoblasts withdrew from the cell cycle and committed to terminal differentiation, DHFR mRNA levels and DHFR synthesis rates decreased with closely matched kinetics. After 15 to 24 h, committed cells contained 5% the proliferative level of DHFR mRNA (80 molecules per committed cell) and synthesized DHFR protein at 6% the proliferative rate. At no point during the commitment process did the decrease in DHFR synthesis rate exceed the decrease in DHFR message. The decrease in DHFR mRNA levels during commitment was sufficient to account fully for the decrease in rates of DHFR synthesis. Furthermore, DHFR mRNA remained polysomal, and the average number of ribosomes per message remained constant (five to six ribosomes per DHFR mRNA). The constancy of polysome size, along with the uniform rate of DHFR synthesis per message, indicated that DHFR mRNA was efficiently translated in postreplicative cells. The results support a model wherein replication-dependent changes in DHFR synthesis rates are determined exclusively by changes in DHFR mRNA levels.

Sequence-independent assembly of spermatid mRNAs into messenger ribonucleoprotein particles.

During mammalian spermatogenesis, meiosis is followed by a brief period of high transcriptional activity. At this time a large amount of mRNA is stored as messenger ribonucleoprotein (mRNP) particles. All subsequent processes of sperm maturation occur in the complete absence of transcription, primarily using proteins which are newly synthesized from these stored mRNAs. By expressing transgene mRNAs in the early haploid spermatids of mice, we have investigated the sequence requirements for determining whether specific mRNAs in these cells will be stored as mRNP particles or be assembled into polysomes. The results suggest that mRNAs which are transcribed in spermatids are assembled into mRNP particles by a mechanism that acts independently of mRNA sequence. Our findings reveal a fundamental similarity between the mechanisms of translational control used in spermatogenesis and oogenesis.

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions.

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.

Early steps in hematogenous metastasis of B16F1 melanoma cells in chick embryos studied by high-resolution intravital videomicroscopy.

BACKGROUND: There are few techniques that permit direct observation of tumor metastasis. The ability to observe steps in this process as they occur in experimental animals would complement studies on molecular mechanisms. PURPOSE: We have developed a novel procedure using high-resolution intravital videomicroscopy to permit direct observation of cells as they arrest in the microcirculation, extravasate, and form micrometastases. We used this procedure to study early steps in experimental metastasis in immune-deficient chick embryos, permitting us to develop this technique in a relatively accessible respiratory organ and in the absence of host immune responses. Our goals were to develop techniques applicable to this host and to other hosts and to clarify the process of hematogenous tumor spread in this host. METHODS: We injected fluorescently labeled B16F1 melanoma cells into the circulation of 11- to 13-day chick embryos, and using intravital videomicroscopy, we observed the cells in the chorioallantoic membrane over time. RESULTS: The majority of injected cells were trapped initially in orifices to the chorioallantoic membrane capillary plexus or in tapering ends of arterioles leading to the plexus. During the first 2 hours, cells were found only in vessel lumina. After 8 hours, 83% of cells had extravasated, and the rest were in the process of extravasation. Cell shape changes and pseudopodial extensions were seen during extravasation and tumor development. Tumor cell division was seen only after extravasation. Tumors tended to develop near microvessels and were often wrapped around them. CONCLUSIONS: Intravital videomicroscopy can provide new information about steps in metastasis. This procedure is applicable to other hosts and can be used in future studies to test hypotheses about molecular mechanisms of tumor spread.

CDKN2 (p16/MTS1) gene deletion or CDK4 amplification occurs in the majority of glioblastomas.

Forty-six glioblastomas, 16 anaplastic astrocytomas, and 8 astrocytomas were studied for the loss of the CDKN2 (p16/MTS1) gene on 9p. The CDKN2 locus was homozygously deleted in 19 of 46 glioblastomas (41%) and 1 allele was lost in an additional 13 cases (28%). The deleted regions were limited centromerically in some cases by the MTS2 locus and telomerically by the 1063.7 locus. CDKN2 was homozygously deleted in 3 of 16 anaplastic astrocytomas (19%) and 2 further cases showed loss of 1 allele. Amplification of the CDK4 gene was present in 7 of 14 (50%) glioblastomas and 3 of 11 (27%) anaplastic astrocytomas with no losses at the CDKN2 locus as well as in 2 of 32 (6%) glioblastomas with CDKN2 losses. Thus one or more of these two genes were shown to be aberrant in 85% of glioblastomas and 50% of anaplastic astrocytomas. None of the 8 astrocytomas showed abnormalities of these genes.

Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations.

The MDM2 (murine double minute 2) gene has recently been shown to code for a cellular protein that can complex the p53 tumor suppressor gene product and inhibit its function. We studied a series of 157 primary brain tumors and report here that the MDM2 gene is amplified and overexpressed in 8-10% of glioblastomas and anaplastic astrocytomas. Thus, MDM2 represents the second most frequently amplified gene after the epidermal growth factor receptor gene in these tumor types. Sequencing of the p53 transcripts in the cases with MDM2 amplification revealed no mutations and restriction fragment length polymorphism analysis showed, with one exception, no losses of alleles on chromosome 17. Our results indicate that amplification and overexpression of MDM2 may be an alternative molecular mechanism by which a subset of human malignant gliomas escapes from p53-regulated growth control.

A common region of homozygous deletion in malignant human gliomas lies between the IFN alpha/omega gene cluster and the D9S171 locus.

Deletions of the 9p-localized type-I interferon (IFN) genes and adjacent loci often occur during the development of malignant glioma. We have applied restriction fragment length polymorphism and microsatellite analysis to 12 loci covering this region of 9p and 3 loci on 9q in 74 human glial tumor tissues to define and further localize the smallest region of hemizygous or homozygous deletion common to the tumors. Three regions of homozygous deletion were evident among the panel of tumors; only one of these, however, residing between D9S171 and the IFN alpha/omega gene cluster, was involved in multiple cases (13 glioblastomas). Hemizygous deletion of this same region was observed in an additional 27 tumors. In total these data indicate the frequent inactivation of a novel tumor suppressor gene residing adjacent to and centromeric of the type-I IFN genes in malignant gliomas.

Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for human astrocytic gliomas with G1-S transition control gene abnormalities.

Deregulation of G1-S transition control in cell cycle is one of the important mechanisms in the development of human tumors including astrocytic gliomas. We have previously reported that approximately two-thirds of glioblastomas (GBs) had abnormalities of G1-S transition control either by mutation/homozygous deletion of RB1 or CDKN2A p16INK4A), or amplification of CDK4 (K. Ichimura et al., Oncogene, 13: 1065-1072, 1996). However, abnormalities of G1-S transition control genes may induce p53-dependent apoptosis in cells. Recent investigations suggest that p14ARF is induced in response to abnormal cell cycle entry and results in p53 accumulation by inhibiting MDM2-mediated transactivational silencing and degradation of p53. To investigate the roles of the G1-S transition control system and the p14ARF/MDM2/p53 pathway in the development of astrocytic gliomas, we examined abnormalities of genes involved in these regulatory pathways in a total of 190 primary human astrocytic gliomas of different malignancy grades [136 GBs, 39 anaplastic astrocytomas (AAs) and 15 astrocytomas (As)]. Sixty-seven percent of GBs (91/136) and 21% of AAs (8/39) had abnormalities of the G1-S control system either by mutation/homozygous deletion of RB1, CDKN2A or CDKN2B, or amplification of CDK4. Seventy-six percent of GBs (103 of 136), 72% of AAs (28 of 39), and 67% of As (10 of 15) had deregulated p53 pathway either by mutation of TP53, amplification of MDM2, or homozygous deletion/mutation of p14ARF. When all of the data were combined and compared, 96% of GBs (87 of 91) and 88% of AAs (7 of 8) with abnormal G1-S transition control also had deregulated p53 pathway. Thus, we demonstrate that deregulation of the G1-S transition control system was almost always accompanied by inactivation of the p53 pathway, clearly illustrating the cooperative roles of these two systems in the development/progression of primary human astrocytic gliomas.

Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver.

It is now known that members of the selectin and integrin families are critical in the initial interaction of cells in circulation with endothelial surfaces. Also, platelet/endothelial cell adhesion molecule-1 has been shown to be involved in transendothelial migration of extravasating cells. Little is known about adhesion molecules involved in subsequent postextravasation events. In this study, the significance of VLA-2 (alpha2beta1) integrin in the movement of human rhabdomyosarcoma RD cells in the liver was characterized by in vivo videomicroscopy. Results show that after extravasation, the mock-transfected RDpF cells were able to migrate to the subcapsular region of the liver. Although the RDX2C2 transfectant expressing VLA-2 integrin extravasated equally well, a majority of RDX2C2 cells remained in close proximity to blood vessels and failed to reach the subcapsular region. The functional involvement of VLA-2 in affecting the ability of RD cells to reach the subcapsular region was verified by the preparation of an RD transfectant [RDX2C2(I-)] expressing a nonfunctional variant of VLA-2 lacking the inserted (I)-domain of alpha2 subunit. In vivo microscopy showed that RDX2C2(I-) cells migrated in a manner similar to control RDpF cells. To demonstrate that RDX2C2 cells that remained in dose proximity to blood vessels were due to VLA-2 function, a blocking monoclonal antibody against VLA-2 (BHA2.1) was prepared. Mice were injected with BHA2.1 or control monoclonal antibody P3 at the time when RDX2C2 cells completed their extravasation. Treatment with BHA2.1 increased the number of RDX2C2 cells that reached the subcapsular region and subsequently formed tumor foci. Therefore, VLA-2 integrin expression has major roles in postextravasation movement and affects tumor foci formation at the liver surface.

Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth.

It is widely accepted that a major role of matrix metalloproteinases in the metastatic process is degradation of basement membrane during cancer cell invasion. We tested the hypothesis that the reduction in metastatic potential which has been demonstrated for B16F10 melanoma cells genetically engineered to overexpress tissue inhibitor of metalloproteinase-1 (TIMP-1) is caused by a decrease in their ability to extravasate. Using intravital videomicroscopy of chick embryo chorioallantoic membrane, we studied extravasation of B16F10 cells and B16F10 cells transfected to overexpress TIMP-1. More than 800 cells in 36 chick embryos were analyzed for each cell line during 72 h postinjection. TIMP-1 upregulation had no effect on the time course of extravasation, virtually all cells from both cell lines having extravasated by 36 h. We also studied the morphology of micrometastases at days 3 and 7. Lack of contact between cancer cells within micrometastases at day 3 and reduction in size and number of tumors at day 7 were observed for TIMP-1 overexpressor cells compared to B16F10. Our findings illustrate that the imbalance between TIMP and metalloproteinases created by overexpression of TIMP-1 in B16F10 cells reduces their metastatic ability in vivo by affecting tumor growth postextravasation.

Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate.

Metastasis is an inefficient process; only a few cancer cells are able to form tumors after being released into the circulation. We studied the fate of cancer cells after injection into the circulation, quantifying their survival and ability to extravasate by 1 day later. B16F10 cells, parental or transfectants overexpressing tissue inhibitor of metalloproteinases 1, were injected i.v. into chorioallantoic membrane of chick embryos and analyzed by intravital videomicroscopy. Cell survival was quantified in two ways: (a) 15-microns microspheres were injected with cancer cells, and proportions of viable cells to microspheres were compared before and after injection; and (b) individual cancer cells were monitored continuously for 0.5-8-h intervals covering the first 24 h. Both methods showed virtually no destruction of cells. Greater than 80% of injected cells survived and extravasated by 24 h, indicating that growth after extravasation is a key stage of metastatic control.

Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency.

Cancer metastasis is an inefficient process. The steps in metastasis responsible for this inefficiency and how metastatic inefficiency can vary in different locations within an organ remain poorly understood. B16F10 cells were injected to target mouse lung, and at sequential times thereafter we quantified in lung the time course of: (a) overall cell survival and metastatic development; and (b) local cell survival and growth with respect to the lung surface and specific interior structures. We found high rates of initial survival of cells trapped in the lung circulation, extravasation into lung tissue, and subsequent survival of extravasated solitary cells (74% at day 3) before metastasis formation. However, at the time of initial replication of metastatic cells a major loss of cells occurred. Although only a small proportion of injected cells started to form metastases, most of these developed into macroscopic tumors. Solitary cells found at later times were dormant. Thus, overall metastatic inefficiency was largely due to postextravasation events affecting solitary cells. Regionally within the lung, cells and metastases were randomly distributed to day 4, but by day 10 preferential tumor growth was found along the lung surface and around arterial and venous vessels. Thus, trapping and early growth of injected cells was unaffected by location within the lung, whereas subsequent metastatic growth was enhanced in specific microenvironments. This study: (a) quantifies early temporal and spatial progression of metastasis in lung; (b) documents persistence of solitary dormant cells; and (c) shows that metastatic inefficiency depends on the initiation of growth in a subset of extravasated cells, whereas continued growth of metastases occurs preferentially in specific tissue environments.

Human glioblastomas with no alterations of the CDKN2A (p16INK4A, MTS1) and CDK4 genes have frequent mutations of the retinoblastoma gene.

A series of 195 human gliomas were studied as to the status of their CDKN2A, CDK4 and RB1 genes. Among 120 glioblastomas, 40% had no wild-type CDKN2A gene, 12% amplified the CDK4 gene, and 14% had no wild-type RBI gene. With two exceptions, each tumour had only one of these abnormalities. Thus the majority of the glioblastomas (64%) had distinct genetic aberrations which would obviously disrupt the control of transition from G1 to the S-phase of the cell cycle. A further 30% had loss of one allele of the CDKN2A and/or RBI genes. Only seven (6%) glioblastomas had no abnormalities of these genes. Anaplastic astrocytomas showed similar changes to the glioblastomas but at lower frequencies-34% showing no aberrations of the genes analysed. The astrocytomas showed solely loss of one allele of the RBI gene in 28% of tumours, with retention of one wild-type copy. In the glioblastomas with no alterations of CDKN2A, CDK4 or RB1, several other genes (CCND1, CCND2, CCND3, CDK6, E2F, CDK7, MYC and MYCN) whose products take part in cell cycle regulation were examined. No abnormalities were detected. Thus some aberration of the CDKN2A, CDK4 and RB1 genes appears to be almost obligatory in glioblastomas.

Clinical targets for anti-metastasis therapy.

Metastasis is responsible for most cancer deaths. Therapeutic strategies to prevent development of metastases thus have potential to impact on cancer mortality. Development of these therapies requires a better understanding of the biology and molecular events of the metastatic process. Metastasis is usually defined, clinically and experimentally, by evidence of the endpoint of the process, that is, the presence of metastatic tumors. Endpoint assays are suitable for determining if a therapeutic approach is effective, but can provide little information on how a treatment works in vivo and what steps in metastasis are affected. We describe here two methodological advances in the study of metastasis as a process: in vivo videomicroscopy, which permits direct observation of steps in metastasis, and a "cell accounting" technique that permits quantification of the fate of cells over time. These procedures have provided new and unexpected insights into the biology of the metastatic process. Based on these insights, we consider which steps in the metastatic process are biologically and clinically most appropriate as therapeutic targets for development of anti-metastasis therapies. We conclude that the most promising stage of the metastasis process for therapeutic targeting is the growth phase, after cancer cells have arrested in the microcirculation in secondary sites and have completed extravasation. Earlier phases in the process are either biologically inappropriate or clinically inaccessible, except in specific cases (e.g., chemoprevention strategies). The role of "seed" and "soil" in determining organ-specific metastasis is also discussed. The metastatic growth phase fortunately is a clinically broad target, and any treatment that limits growth of metastases prior to their causing irreversible harm to the patient has the potential to be clinically useful. A variety of therapeutic approaches to target this phase are under active development, including inhibition of angiogenesis or signal transduction pathways needed to support the growth of metastatic cells.

Oligodendroglial tumors frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes.

We investigated 34 oligodendroglial tumors (7 oligodendrogliomas, 11 anaplastic oligodendrogliomas, 8 oligoastrocytomas, and 8 anaplastic oligoastrocytomas) for deletion, mutation, hypermethylation, and expression of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes at 9p21. One anaplastic oligoastrocytoma carried a homozygous deletion including all 3 genes. None of the tumors demonstrated point mutations in any of the genes. Methylation-specific polymerase chain reaction (MSP) analysis and sequencing of bisulfite-modified DNA, however, revealed frequent hypermethylation of the 5'-CpG islands in CDKN2A, p14ARF, and CDKN2B. Partial or complete methylation of the majority of CpG sites analyzed from each gene was detected in 32% of the tumors at the CDKN2A gene and at a similar percentage (41%) of the tumors at the p14ARF gene and the CDKN2B gene. Most tumors with CDKN2A, p14ARF, and/or CDKN2B hypermethylation either lacked detectable transcripts from these genes or had lower mRNA levels than those determined for non-neoplastic brain tissue. There was a significant correlation between hypermethylation of these genes and the presence of allelic losses on chromosomal arms 1p and 19q. In addition, p14ARF hypermethylation was predominantly found in tumors without a demonstrated TP53 mutation. Taken together, our results indicate that hypermethylation of CDKN2A, p14ARF, and CDKN2B is an important epigenetic mechanism by which oligodendroglial tumors may escape from p53- and pRb-dependent growth control.