A. I. Protopopov

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma

Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22–23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.

TSLC1 Is a Tumor Suppressor Gene Associated with Metastasis in Nasopharyngeal Carcinoma

In up to 87% of nasopharyngeal carcinoma (NPC) clinical tumor specimens, there was either down-regulation or loss of TSLC1 gene expression. Using a tissue microarray and immunohistochemical staining, the frequency of down-regulated or loss of expression of TSLC1 in metastatic lymph node NPC was 83% and the frequency of loss of expression of TSLC1 was 35%, which was significantly higher than that in primary NPC (12%). To examine the possible growth-suppressive activity of TSLC1 in NPC, three NPC cell lines, HONE1, HNE1, and CNE2, were transfected with the wild-type TSLC1 gene cloned into the pCR3.1 expression vector; a reduction of colony formation ability was observed for all three cell lines. A tetracycline-inducible expression vector, pETE-Bsd, was also used to obtain stable transfectants of TSLC1. There was a dramatic difference between colony formation ability in the presence or absence of doxycycline when the gene is shut off or expressed, respectively, with the tetracycline-inducible system. Tumorigenicity assay results show that the activation of TSLC1 suppresses tumor formation in nude mice and functional inactivation of this gene is observed in all the tumors derived from tumorigenic transfectants. Further studies indicate that expression of TSLC1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)-G(1) phase in normal culture conditions, whereas in the absence of serum, TSLC1 induced apoptosis. These findings suggest that TSLC1 is a tumor suppressor gene in NPC, which is significantly associated with lymph node metastases.

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.

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.

Human chromosome 3: integration of 60 NotI clones into a physical and gene map

Sequence tagged sites generated for 60 NotI clones (NotI-STSs) from human chromosome 3-specific NotI-jumping and NotI-linking libraries were physically located using PCR screening of a radiation hybrid (RH) GeneBridge4 panel. The NotI map of chromosome 3 was generated using these RH-mapping data and those obtained earlier by FISH and sequencing of the corresponding NotI clones. The sequences of the NotI clones showed significant homologies with known genes and/or ESTs for 58 NotI-STSs (97%). These 58 NotI clones displayed 91–100% identity to 54 genes and 23 cDNA/EST clones. One known and two hypothetical protein-coding genes were localized for the first time and nine cDNA clones (unknown genes) were also carefully mapped only in this work. Three newly mapped genes are histone gene H1X (NR1-BK20C) and genes for hypothetical proteins THC1032178 and THC1024604 (NL1-243).

Human NRG3 gene Map position 10q22-q23

Neuregulin (NRG3) is a novel, neural-enriched ligand for ErbB4. Murine NRG3 (AF10130) is predicted to contain an extracellular domain with an EGF motif, a transmembrane domain, and a large cytoplasmic domain. The expression of NRG3 is highly restricted to the developing and adult nervous system [1]. Recombinant NRG-3 altered the growth of human breast cancer cells growing in vitro [2]. BLASTX search revealed that a partial sequence of our NotI linking clone NR5-100 (7.5 kb insert, sequence accession No. AJ277142, 305 bp length, [3]) possess 95% similarity over all sequence to mouse NRG3 gene and displays 98% identity in 100aa overlap. Sequences for the human NRG3 are not available but NR5-100 has 99% identity to human DNA sequence No. AL096706 (chromosome 10, sequencing in progress). Taking into account the level of sequencing errors we suggest that NR5-100 contains at least part of the human NRG3 gene. Detailed protocols for preparing metaphase cells, £uorescence in situ hybridization, and signal analysis were as described earlier [4]. Twenty-eight metaphases from a normal male all showed speci¢c labeling of 10q22-q23. Four chromatids were labeled in 14 cells, 3 chromatids in 20 cells and 2 chromatids in 4 cells. No additional signals were found.

Assignment1 of CDK5R2 coding for the cyclin-dependent kinase 5, regulatory subunit 2 (NCK5AI protein) to human chromosome band 2q35 by fluorescent in situ hybridization

Neuronal CDC2-like kinase (OMIM: 116940) is a heterodimer of CDK5 (OMIM: 123831) and p25 (nck5a), a neuronspecific 25-kDa regulatory subunit derived proteolytically from NCK5A, encoded by CDK5R1 (neuronal CDK5 activator or cyclin-dependent kinase 5 regulatory subunit 1 gene) (Lew et al., 1994; Tsai et al., 1994). By screening a human hippocampus library with a bovine Nck5a cDNA, Tang et al. (1995) isolated cDNAs encoding NCK5AI, a distinct NCK5A isoform. They also referred to the protein as p39 (nck5ai) based on its calculated molecular mass of 39 kDa. This isoform showed a high degree of sequence similarity to p35 (NCK5A) with 57% amino acid identity. A 30-kDa truncated form of p39 (nck5ai) activated both recombinant and native CDK5 in vitro, as does p25 (nck5a). Northern blot analysis of rat tissues indicated that both NCK5A and p39 (nck5ai) are expressed exclusively in brain. In situ hybridization to rat brain sections revealed that p39 (nck5ai) mRNA was highly expressed in the CA1 to CA3 zone of the hippocampal formation, an area highly enriched in neurons. There was no expression in the fimbria hippocampi, where glial cells predominate. Tang et al. (1995) concluded that p39 (nck5ai) shares many common characteristics with NCK5A, including CDK5-activating activity and brainand neuron-specific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins. The gene encoding p39 (nck5ai) was called CDK5R2. Partial sequencing of the NotI linking clone NR3-007 (Zabarovsky et al., 1994) revealed that it is 100% identical to CDK5R2 over 378 bp (aa 110–235). We concluded that NR3-007 contains part of CDK5R2 and mapped this gene using the NR3-007 clone.

Human ALY/BEF gene Map position 17q25.3.

Recently a human gene coding for the nuclear protein that dramatically increases DNA binding of transcription factors containing a basic leucine zipper (bZIP) DNA-binding domain has been cloned (AF047002) [1,2]. This gene was called transcriptional coactivator ALY [1] or bZIPenhancing factor BEF [2]. The ALY plays a role in regulating the activity of TCRa enhancer complex and functions as a molecular chaperone. ALY/BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers; the elevated concentration of the bZIP dimer then drives the DNA binding reaction. Clone NR1-OD19 (insert size 7 kb, GenBank Accession No. AJ277143) was isolated from human NotI linking library [3] and partially sequenced. The NCBI BLAST analysis revealed that this clone is 100% identical to the human ALY/BEF gene over 134 bp (56^99 aa). Thus we concluded that our NR1-OD19 contains part of the ALY/BEF gene. In the present study we mapped the ALY/BEF gene using NR1-OD19 clone as a probe. Detailed protocols for preparing metaphase cells, £uorescence in situ hybridization, and signal analysis were as described earlier [4]. Forty metaphases from a normal male all showed speci¢c labeling of distal 17q25.3, on all 4 chromatids in 11 cells, 3 chromatids in 19 cells and 2 chromatids in 11 cells. No additional signals were found.

Human SS13 gene Map position 17q25.3.

The SSI3 (STAT-induced STAT inhibitor-3, NM___003955), also called SOCS3 or CIS3 [1^3] is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity and thereby suppressing cytokine signalling [1]. The SSI3 gene encodes a deduced 225-amino acid protein [1]. Song and Shuai (1998) found that SOCS3 inhibits IFN-mediated Janus-activated kinase/STAT signalling pathways [4]. Transgene-mediated expression in mice blocked fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion in mice resulted in an embryonic lethality at 12^16 days associated with marked erythrocytosis [5]. Partially sequenced NotI linking clone NL1-BJ17 (insert size 5.7 kb, GenBank Accession No. AJ29043, 576 bp length) revealed that this clone shows 99% identity to the SSI3 in a 575 bp overlap, corresponding to protein region 1^168 aa. Thus we concluded that NL1-BJ17 contains at least part of SSI3 and used this clone for the mapping of SSI3. FISH was done as previously described [6]. Forty-three metaphases from a normal male all showed speci¢c labelling of 17q25.3. Four chromatids were labelled in 17 cells, 3 chromatids in 8 cells and 2 chromatids in 18 cells. No background signals were found.

Human COP9 subunit 8 homolog gene SGN8 Map position 2q37.

The COP9 is conserved from plants, ¢ssion yeast, and insects to human cells [1^3]. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways. Disruption of COP9 signalosome in D. melanogaster caused lethality [3]. Human COP9 homolog is ubiquitously expressed and involved in signal transduction. Partially sequenced NotI linking clone NR1-OC6 (insert size 4.5 kb, GenBank accession No.AJ277144) [4] revealed that this clone shows 99% identity to the hCOP9 in a 129 bp overlap (single nucleotide change), corresponding to protein region 1-43 aa. Thus we concluded that NR1-OC6 contains at least part of hCOP9 and used this clone for the mapping of hCOP9. FISH was performed as previously described [5]. Twenty-seven metaphases from a normal male all showed speci¢c labelling of 2q37. Four chromatids were labelled in 10 cells, 3 chromatids in 13 cells and 2 chromatids in 4 cells. No background signals (sites with >2 signals) were found. This data con¢rm one of the previous localization of hCOP9 (it was mapped between D2S2158 and D2S125, genetic size of bin 33 cM).