The p16INK4a/CDKN2A tumor suppressor and its relatives

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin’s growth-inhibitoryactivitydepends on specific interaction with the cy toplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

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The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

Hypoxia poses many problems to the treatment of cancer. Hypoxic tumors are more resistant to chemotherapy and radiation. In addition, hypoxia induces a number of genes responsible for increased invasion, aggressiveness, and metastasis of tumors. The augmented metastatic potential due to hypoxia-mediated gene expression is discussed in this section. Particular attention is given to recent studies of specific genes involved in the key steps of metastasis, including extracellular matrix interactions, migration, and proliferation.

Hypoxia, gene expression, and metastasis

p16(MTS-1/CDKN2/INK4a) in cancer progression.

Bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), enamelin (ENAM), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN) are glycophosphoproteins expressed in bones and/or teeth. Direct comparison of their amino acid sequences do not suggest that they belong to a single genetic family, but a detailed analysis of their chromosomal location and gene structure does. Analysis of human brain mRNA by RT-PCR has led to the discovery of two additional exons thereby making it more convincing that MEPE is a member of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family. We propose that the members of this SIBLING family are extended, flexible proteins in solution that can facilitate the formation of a number of different complexes. For example, OPN can bridge complement Factor H to either an RGD-dependent integrin or to CD44 forming a membrane-bound complex that actively suppresses the alternate complement pathway. Two possible mechanisms for inhibiting the lytic pathway of alternate complement are presented.

Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins.

Expression of osteopontin in human glioma. Its correlation with the malignancy.

✓ Expression of monocyte chemoattractant protein-1 (MCP-1) in human glioma cell lines and surgical specimens was studied by Northern blot analysis, reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The samples tested consisted of 11 human glioma cell lines and eight specimens of human malignant glioma (seven from glioblastomas and one from a malignant ependymoma). Messenger ribonucleic acid (mRNA) of MCP-1 was detected by either Northern blot or reverse-transcription polymerase chain reaction analysis in all cell lines and tumor specimens examined. In vivo expression of MCP-1 mRNA and protein was found predominantly in glioma cells with large and pleomorphic nuclei rather than in areas of small nucleated glioma cells. Adjacent brain tissue did not produce a significant level of MCP-1 mRNA or protein. Tumor vessels with endothelial proliferation expressed a moderate level of MCP-1 protein. Macrophages were found among the glioma cells, and the degree of macro...

Expression and localization of messenger RNA and protein for monocyte chemoattractant protein-1 in human malignant glioma

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.

In the present study we investigated the frequency of p16 gene exon 2 mutations in 35 malignant gliomas, using either direct sequencing of the PCR products or cloning into the pCRII vector and sequencing of the cloned PCR products. No mutations were detected during direct sequencing of the PCR products. However, after sequencing of individual clones, we found multiple mutations in 5 tumors involving codons 73(GCC to ACC, Ala to Thr), 76 (GCC to GTC, Ala to Val), 85(GCT to ACT, Ala to Thr), 98(CAC to TAC, His to Tyr), 102 (GCG to GTG, Ala to Val), 106 (GTG to ATG, Val to Met), 107 (CGC to TGC, Arg to Cys), 127 (GCA to GTA, Ala to Val), 128 (CGG to TGG, Arg to Trp) and 136 (GGC to GAC, Gly to Asp). Mutations were found only in glioblastomas and were either C to T or G to A transitions. Each mutation was detected in a small percentage of tumor cells (1.3-22%) using individual colony sequencing and southern hybridization with mutant oligonucleotides, consistent with the heterogenous cell population of glioblastomas. The presence of p16 gene mutations only in glioblastomas suggests that they are late events in glioma development.

Mutations of the p16 gene in gliomas.

Background: Primary central nervous system lymphoma is an extranodal form of non-Hodgkin lymphoma arising in the craniospinal axis. The incidence of PCNSL is reportedly on the increase in some parts of the world in the last 2 decades. Methods: We surveyed 4007 patients diagnosed with PIT between 1989 and 2004, with histologic diagnosis being obtained in 70% of the patients. Of these, the PCNSL cases were reviewed. Results: Of 4007, 136 (3.4%) carried a diagnosis of PCNSL. At 0.41 per 100,000 per year, the ageadjusted incidence rate for that period was higher than that for the period from 1989 to 1998 (0.29 per 100,000 per year). Moreover, the number of patients with PCNSL doubled from 45 (1989-1996) to 91 (1997-2004). Conclusions: Our findings point to an increase in the incidence of PCNSL among immunocompetent individuals in Kumamoto Prefecture.

Hideo Takeshima

Papers

Expression of osteopontin in human glioma. Its correlation with the malignancy.

Expression and localization of messenger RNA and protein for monocyte chemoattractant protein-1 in human malignant glioma

Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.

Mutations of the p16 gene in gliomas.

Neoplasm Rising incidence of primary central nervous system lymphoma in Kumamoto, Japan