Functional Characterization of the Candidate Tumor Suppressor Gene NPRL2/G21 Located in 3P21.3C

[CANCER RESEARCH 64, 6438–6443, September 15, 2004] Functional Characterization of the Candidate Tumor Suppressor Gene NPRL2/G21 Located in 3p21.3C

Jingfeng Li,1 Fuli Wang,1 Klas Haraldson,1 Alexey Protopopov,1 Fuh-Mei Duh,2,3 Laura Geil,2,3 Igor Kuzmin,2,3 John D. Minna,4 Eric Stanbridge,5 Eleonora Braga,6 Vladimir I. Kashuba,1,7 George Klein,1 Michael I. Lerman,2 and Eugene R. Zabarovsky1,8 1Microbiology and Tumor Biology Center, Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden; 2Cancer-Causing Genes Section, Laboratory of Immunobiology, Center for Cancer Research, National Cancer Institute, Frederick, Maryland; 3Basic Research Program, SAIC-Frederick, Inc., Frederick, Maryland; 4Hamon Center for Therapeutic Oncology, Research, University of Texas Southwestern Medical Center, Dallas, Texas; 5Department of Microbiology and Molecular Genetics, College of Medicine, University of California at Irvine, Irvine, California; 6Russian State Genetics Center, Moscow, Russia; 7Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine; and 8Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

ABSTRACT accompanied by chromosome 3p homozygous deletions, is a charac- teristic feature of most major epithelial carcinomas, such as lung, Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the breast, cervical, oral cavity, ovary, and kidney (2, 3). These changes lung cancer region, as a strong candidate tumor suppressor gene. Here we indicate the involvement of multiple tumor suppressor genes. provide additional evidence of the tumor suppressor function of NPRL2/ G21. The gene has highly conserved homologs/orthologs ranging from We have performed a comprehensive deletion survey of 3p on more yeast to humans. The yeast ortholog, NPR2, shows three highly conserved than 400 major epithelial carcinoma cases (4–8), using a defined set regions with 32 to 36% identity over the whole length. By sequence of markers, combining conventional loss of heterozygosity with quan- analysis, the main product of NPRL2/G21 encodes a soluble protein that titative real-time PCR, and comparative genomic and Southern hy- has a bipartite nuclear localization signal, a protein-binding domain, bridizations. We identified two most frequently affected 3p21.3 re- similarity to the MutS core domain, and a newly identified nitrogen gions, LUCA at the centromeric and AP20 at the telomeric border of permease regulator 2 domain with unknown function. The gene is highly 3p21.3. Aberrations of either region were detected in more than 90% expressed in many tissues. of the studied tumors. These 3p21.3 aberrations were complex and, in We report inactivating mutations in a variety of tumors and cancer cell addition to deletions, could involve gene amplifications as well. lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 Homozygous deletions were detected in 10 to 18% of all of the tumors 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 in both the LUCA and AP20 sites. The frequent chromosome aber- showed homozygous deletions in the 3؅ end of NPRL2 in 2 renal, 3 lung, rations in these regions suggest that they harbor multiple tumor .(and 1 cervical (HeLa) cell line. Deletions in the 3؅ part of NPRL2 could suppressor genes (2, 3 result in improper splicing, leading to the loss of the 1.8 kb functional Analysis of 15 homozygous deletions in the LUCA region permit- NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be ted us to establish the smallest homozygously deleted region in involved in mismatch repair, cell cycle checkpoint signaling, and activa- 3p21.3C, located between D3S1568 (CACNA2D2 gene) and D3S4604 tion of apoptotic pathway(s). The yeast NPR2 was shown to be a target of (SEMA3F gene). It contains 17 genes that were defined previously as cisplatin, suggesting that the human NPRL2/G21 may play a similar role. lung cancer candidate tumor suppressor genes. Mapping of 19 ho- At least two homozygous deletions of NPRL2/G21 were detected in 6 mozygous deletions in the 3p21.3T/AP20 region resulted in localiza- tumor biopsies from various locations and with microsatellite instability. tion of its smallest homozygous deletion to the region flanked by This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene. D3S1298 and D3S3623. It contains 4 genes, namely APRG1, ITGA9, RBSP3/HYA22, and VILL, which need to be analyzed (9). In this study, we report the functional characterization of the INTRODUCTION NPRL2/G21 gene from the LUCA region and its tumor suppressor Epithelial tumors are the most prevalent and lethal cancers in the genes-like features. world. For example, lung cancer alone kills Ͼ150,000 patients each year in the United States and more than a million around the world (1). MATERIALS AND METHODS Loss of heterozygosity involving several chromosome 3p regions, Cell Lines and General Methods. Small cell lung carcinoma cell line U2020 was described earlier (10). The ACC-LC5 small cell lung carcinoma Received 12/10/03; revised 6/1/04; accepted 7/9/04. Grant support: the Swedish Cancer Society, the Swedish Research Council, the cell line that carried a deletion in 3p21.3 (11) was kindly provided by Dr. Swedish Foundation for International Cooperation in Research and Higher Education Yusuke Nakamura (University of Tokyo, Tokyo, Japan), and the GLC20 small (STINT), the Swedish Institute, the Royal Swedish Academy of Sciences, INTAS, cell lung carcinoma cell line with homozygous deletion in LUCA region was Karolinska Institute, The Program of Russian Academy of Science Basic Research - to obtained from Dr. Charles H. Buys (12). A498, Caki1, and Caki2 renal clear Medicine, the Russian Ministry of Science and Technology, the Russian Foundation for Basic Research Grant 01-04-48028 (E. Braga), the National Cancer Institute, NIH contract cell carcinoma lines, 7 cervical carcinoma cell lines (HTB, C33A, HeLa, no. NO1-CO-56000 (M. Lerman), NIH contract no. NO1-CO-12400 (F. Duh, L. Geil, and CaSki, C4–1, MS-75, and SiH2), and the N417 small cell lung carcinoma cell I. Kuzmin), Lung Cancer SPORE P50 CA70907 and CA71618 (J. Minna), and the Avon line were purchased from the American Type Culture Collection (Manassas, Foundation (E. Stanbridge). MD). Lymphoblastoid cell line CBMI-Ral-STO, osteosarcoma cell line The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Saos-2, non-small cell lung carcinoma line A549, and renal clear cell carci- 18 U.S.C. Section 1734 solely to indicate this fact. noma lines KRC/Y, ACHN, TK164, HN4, TK10, and KH-39 were obtained Note: The content of this publication does not necessarily reflect the views or policies from the MTC-KI (Stockholm, Sweden) cell lines collection (6). of the Department of Health and Human Services, nor does mention of trade names, KRC/Y, Caki 1, Caki 2, ACHN, TK10, and TK164 cell lines were karyo- commercial products, or organizations imply endorsement by the United States Govern- ment; J. Li and F. Wang contributed equally to this work; J. Li is currently in the National typed using trypsin-Giemsa banding, and different cell clones were found (data Cancer Institute, Frederick, Maryland; A. Protopopov is currently in the Dana-Farber not shown). Heterogeneity of the small cell lung carcinoma U2020 and renal Cancer Institute, Boston, Massachusetts. cell carcinoma KH39 cell lines was shown previously (10, 13). Requests for reprints: Eugene R. Zabarovsky, Microbiology and Tumor Biology Molecular cloning of the human NPRL2/G21 gene was described previously Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Phone: 46-8-728- 67-50; Fax: 46-8-31-94-70; E-mail: [email protected]. (ref. 14; accession no. AF040707). The structures of NPRL2 and PCR primers ©2004 American Association for Cancer Research. used in the study are shown in Fig. 1. 6438

Fig. 1. Genomic structure of NPRL2 (A) and primers (B) used in the study. Accession no. for NPRL2 is AF040707; accession no. for BLU is NM_015896; and accession no. for RASSF1A is NM_007182. Noncoding sequences are shown in black. Thick horizontal arrows indicate the orien- tation of transcription. Position of the F2a primer on the RASSF1A gene transcript is 97-116nt from the 5Ј end. Small arrowheads indicate position of PCR primers. (ex, exon)

For amplification of RASSF1A gene, the following primers were used: RESULTS AND DISCUSSION forward, F2a 5Ј-GCCCAAAGCC AGCGAAGCAC-3Ј; and reverse, F2int2 5Ј-ACCCAGGCAG CCCTCGAGAA-3Ј (Fig. 1). The PCR primers were Specific Aims and Experimental Design. The NPRL2 gene was purchased from Invitrogen (Carlsbad, CA). The PCR was performed as de- one of the promising tumor suppressor gene candidates that we scribed earlier (4). However, we used only 28 cycles for detection of homozy- analyzed previously (14). Forced, uncontrolled expression of wild- gous deletions in NPRL2 gene in both cancer cell lines and tumor biopsies. type NPRL2/G21 transgenes from adenovirus vectors inhibited tumor This was done to reduce the effect of normal cell contamination in the case of cell growth by inducing apoptosis and cell cycle arrest of non-small biopsies and heterogeneity in the case of cancer cell lines. cell lung carcinoma line cell lines H1299 and A549. Also, intratu- PCR products were cloned, using the TOPO TA cloning kit for sequencing moral injection of a NPRL2 expressing adenovirus vector or systemic (Invitrogen). All molecular biology and microbiology procedures were performed as administration of protamine-complexed vectors significantly sup- described previously (15, 16). Plasmid DNA was purified using the R.E.A.L.- pressed the growth of H1299 and A549 tumor xenografts and inhib- Prep kit (Qiagen, Valencia, CA). Sequencing was done using an ABI 310 ited A549 experimental lung metastases in nu/nu mice (19). Although Sequencer (Applied Biosystems, Foster City, CA), according to the manufac- suggestive, these results could not be interpreted unequivocally, be- turer’s protocol. Methylation studies were performed as described previously cause adenovirus-mediated overexpression was not controlled and (17). could lead to artificial inhibition of tumor growth. Southern and Northern transfer and hybridization were performed as de- In this study, we reinvestigated the tumor suppressing function of scribed earlier (15, 16). To determine the expression pattern of NPRL2/G21 NPRL2 under more physiologic conditions using the previously de- gene in normal tissues, Northern hybridizations with human multiple tissue #7760-1 Northern blots (Clontech, Palo Alto, CA) were performed. scribed tetracycline-controlled gene inactivation test (18, 20). This Cell and tumor growth assays were done as described previously (17, 18). test is based on the functional inactivation of the analyzed genes in NPRL2/G21 open reading frames from pCR4-TOPO clones (digested by conjunction with tumor growth in SCID mice. Our hypothesis was NcoI and EcoRI, and blunted by Klenow Fragment) were reinserted into that under selective pressure in vivo, the introduced tumor suppressor blunted NheI sites of pETE (elimination test episomal plasmid) vectors (18). genes must be inactivated in growing experimental tumors (by dele- Bioinformatics. DNA homology searches were performed using BLASTX tion, mutation, and promoter methylation) as they are in naturally and BLASTN programs at the National Center for Biotechnology Information growing patient tumors. server. Sequence assembling was done using DNASIS (HITACHI-Pharmacia, Because the LUCA region is a frequent target of hemizygous and Freiburg, Germany). The BEAUTY Post-Processor was used with the homozygous deletions in renal cancer (2, 3, 6, 8), we also analyzed BLASTP protein databases searches provided by the Human Genome Se- quencing Center (Houston, TX).9 Scanning of the PROSITE and the Pfam A NPRL2/G21 for mutations and/or loss of expression in renal cell protein families and domains was performed at the Swiss Institute for exper- carcinoma cell lines. imental Cancer Research10 and at the National Center for Biotechnology Growth Inhibiting Activity of NPRL2 In vitro and In vivo. To Information server (CD-Search).11 Proteins domains were detected by test for the growth suppressing effect of NPRL2/G21, we performed SMART12 and Motif Scan.13 colony formation assays using the KRC/Y renal cell carcinoma cells that were used in our previous studies (3, 17, 18) and showed aberrant 9 Web address: dot.imgen.bcm.tmc.edu: 9331. expression of NPRL2 (see below). Selection was done for expression 10 Web address: www.isrec.isb-sib.ch/software/PFSCAN_form.html. 11 Web address: www.ncbi.nlm.nih.gov/. 12 Web address: smart.embl-heidelberg.de/. 13 Web address: scansite.mit.edu/motifscan_seq.phtml. 6439

mutated RASSF1A didn’t exhibit any effect on cell growth. Fig. 4A shows the results. NPRL2/G21 expression suppressed cell growth in the absence of doxycycline (80 to 90% suppression from day 7). A partial growth inhibition effect was also seen in the presence of doxycycline. Leakage of expression and growth inhibition because of the doxycycline effect (22) are the most important reasons for the slower growth of UG1 cells in the presence of doxycycline. UG1 cells (5 ϫ 106) were inoculated into 10 SCID mice, and U2020 cells were used as a control. Previously, we have shown that Fig. 2. The effect of expression of NPRL2 on colony formation efficiency in KRC/Y tetracycline doesn’t affect the growth of parental cell lines (18, 20, cells. Efficiency of colony formation for the pETE vector is taken as 100%. RASSF1A was 21). Five of the mice were then given 1 mg/ml tetracycline in their Ϯ used as a positive control. Bars, SD. water, whereas the remaining five were not. Strong inhibition of tumor growth was seen in the mice, and only 3 large tumors have of the Hyg gene, carried by the pETE vector. As a negative control, grown (Table 1; Fig. 4B). The one tumor that grew in a tetracycline- the empty pETE vector was used. Transfection with pETE containing consuming SCID mouse exhibited only a slightly delayed growth rate, the RASSF1A gene served as a positive control. Fig. 2 shows that compared with the control U2020 cells. It is known that tetracycline NPRL2/G21 inhibited colony formation similarly to RASSF1A. is a weaker inhibitor of expression compared with doxycycline in the For the gene inactivation test assay, described previously (18, 20, tetracycline transactivator system, and it is likely that expression 21), the pETE-Hyg vector (containing the complete open reading leakage is stronger in vivo in SCID mice than in vitro. Even weak frame for NPRL2) was transfected in the presence of doxycycline into expression of the NPRL2 because of the leakage could have a strong cells constitutively producing the tetracycline transactivator. This growth inhibiting effect. system permitted the modulation of gene expression both in vitro and After 3 weeks, tumors were explanted and tested for the presence of in vivo. The transfected genes are expressed in the absence but not in transgenic NPRL2 by PCR. The transgenic NPRL2 was detected in all the presence of tetracycline or its analog doxycycline. The system three samples. These three cases were additionally examined with simulates physiologic expression levels. Northern hybridization. In tumor T3, no expression was detected; Two tetracycline transactivator-producing cell lines were used: the although good expression was found in T1, and very weak expression non-small cell lung carcinoma line cell line A549 and the small cell was found in T2 (Fig. 5A). The transgenic NPRL2 gene was se- lung carcinoma line U2020. The two clones chosen showed the best quenced in T1 and T2, and in both cases missence mutations were tetracycline regulation (at least 100-fold difference between induced discovered (in T1 Arg324Gly and in T2 His16Arg; see Fig. 5B). It is and repressed state; see example in Fig. 3). For the U2020 cell line, likely that these mutations could have led to faster growth of the T1 pETE-NPRL2 clone 1 (UG1) was chosen and for A549, pETE- and T2 tumors after week 3. NPRL2 clone 1 (AG1). In another experiment, AG1 cells were inoculated into 8 SCID The UG1 clone was assessed for growth on plastic Petri dishes in mice, and no tumors were observed after 8 weeks (Fig. 6). the absence or presence of doxycycline. The original cells were used These experiments extended our previous observations. They dem- as a control because, in our previous papers (see ref. 3, 17, 18, 20, 21), onstrated that NPRL2/G21 has true growth inhibiting activity both in we showed that empty pETE vector, 3PK, MLH1, mutated RB1, and vitro and in vivo, and not only in non-small cell lung carcinoma line but also in small cell lung carcinoma cell lines. Analysis of NPRL2 Gene in Renal Cell Carcinoma Cell Lines. As described in the previous section, NPRL2/G21 strongly inhibited colony formation by KRC/Y cells. This confirmed our hypothesis that loss or inactivation of this gene may be important for the development of renal cell carcinoma and is the reason why we decided to study this gene in renal cell carcinoma lines more carefully. We tested 8 renal cell carcinoma cell lines for the methylation of the 5Ј end of the NPRL2 gene using G21methyF and G21methyR Fig. 3. Dox-dependent NPRL2 expression in vitro for transfected U2020 cell clones UG4 and UG1. Gel stained with ethidium bromide was used for loading control (lower primers (see Fig. 1). Direct bisulfite sequencing analysis did not panel). reveal any methylation.

Fig. 4. Growth inhibition of U2020 cells by NPRL2 (clone UG1) gene in vitro (A) and in vivo (B). A, An average of three independent experiments is presented. B, The average size of 3 tumors for U2020 and of 2 tumors for UG1 without tetracycline (T1 and T2; see Table 1) is given. For UG1 with tetracycline, growth curve for T3 is shown.

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Table 1 Summary of tumor growth inhibition (SCID mice) experiments Gene Clones Tetracycline Tumors PCR Northern Mutation NPRL2 in U2020 clone1(UG1) Ϫ T1 ϩϩAGG to GGG (Arg324Gly) Ϫ No tumor Ϫ T2 ϩϮCAC to CGC (His16Arg) Ϫ No tumor Ϫ No tumor ϩ T3 ϩϪ ϩ No tumor ϩ No tumor ϩ No tumor ϩ No tumor

Then we performed Northern analysis of 7 renal cell carcinoma cell deletion was found in the renal cell carcinoma cell line ACHN (data lines. It is known that NPRL2 is expressed in multiple splice isoforms not shown). We have found a similar situation in lung cancer cell (see Fig. 7A; ref. 14). The main transcript in normal kidney was found lines: the 3Ј part of NPRL2 was completely absent in U2020 and A549 to be ϳ1.8-kb size. However, in all of the 7 tested renal cell carcinoma (Fig. 7C). One of the tested small cell lung carcinoma lines, GLC20, cell lines this transcript was absent, and only a ϳ7-kb transcript was had the whole LUCA region homozygously deleted. One more ho- detected (Fig. 7B). mozygous deletion of the 3Ј part of NPRL2 was detected in the H647 To understand the basis of these changes, we performed PCR with non-small cell lung carcinoma line (data not shown). Interestingly, the genomic DNA using different primers located in the NPRL2 gene (see 3Ј end of NPRL2 was also homozygously deleted in 1 of 7 (HeLa) Fig. 7C and Fig. 1 for location of primers) and found frequent changes cervical carcinoma cell lines. Thus, after screening 7 renal cell car- in the 3Ј part of the gene. In 1 (HN4) of 6 renal cell carcinoma lines cinoma, 3 small cell lung carcinoma (without GLC20), 2 non-small shown in Fig. 7C, the 3Ј part was completely absent. The same cell lung carcinoma, and 7 cervical carcinoma lines, we found the 3Ј end of NPRL2 homozygously deleted in 6 of them. Additionally, homozygous deletions of NPRL2 were found in the prostate cancer cell line LNCaP and oral squamous carcinoma line SCC15 (data not shown). In all of the of these cell lines (except HeLa and LNCaP), the homozygous deletions included the RASSF1A gene (see Fig. 1 and Fig. 7C; F2a-F2int2 primers). In HeLa and LNCaP, the homozygous deletions involved only the 3Ј end of NPRL2 and did not involve the RASSF1 coding sequences. However, we cannot exclude the possi- bility that in these cases RASSF1A was inactivated because of deletion of noncoding or 5Ј regulatory sequences. Deletions in the 3Ј part of NPRL2 gene could result in improper splicing, leading to the loss of the functional 1.8-kb NPRL2 mRNA. Preliminary analysis of expressed sequence tag (EST) databases, as well, revealed the presence of nonsense mutations in NPRL2 clones obtained from tumor cells/ tissues. For instance, deletion of 172-176nt downstream from ATG codon was found in lymphoma (BR061866), neuroblastoma (AL526015), and retinoblastoma (BM413639) cells. A nonsense deletion was found also in small cell lung carcinoma BC008802, and missence mutations were found in uterine (A1884407), colon (BE867114), brain (A1933357, A1199457), and other tumors. Conclusions and Hypothesis. In summary, we found that the NPRL2/G21 gene has growth inhibitory activity for renal cell carcinoma (KRC/Y), small cell lung carcinoma (U2020), and non-small Fig. 5. Analysis of SCID tumors. A, Expression of NPRL2 in xenografts tested with cell lung carcinoma (A549) cell lines when tested under controlled Northern hybridization. Gel stained with ethidium bromide was used for loading control (bottom panel). ϩ, mouse received water with tetracycline. B, mutations in SCID tumors physiologic conditions of gene expression (see ref. 2, 21) both in vitro T1 and T2. The mutated positions of NPRL2 gene (accession no. AF040707) are indicated and in vivo in SCID mice. We have also found mutations in experi- by arrows. mental tumors and intragenic homozygous deletions in renal cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, and other cancer cell lines (cervical HeLa, prostate LNCaP, and oral squamous SCC15). Preliminary analysis of EST databases also revealed the occurrence of nonsense and missense mutations in NPRL2 clones obtained from lymphoma, neuroblastoma, retinoblastoma, uterine, colon, brain, and other tumors. All of these features are consistent with the conclusion that NPRL2/G21 is a multiple tumor suppressor gene. Its inactivation or loss may favor the development of breast and cervical carcinomas, as we have detected previously frequent homozygous deletions affecting the LUCA region in these tumors (7, 8). The NPRL2/G21 gene has highly conserved orthologs in species Fig. 6. Tumor growth inhibition of A549 cells by NPRL2 (clone AG1) in vivo in SCID mice. The average size of 4 tumors is shown for A549, and growth of 8 inoculates of AG1 ranging from yeast to humans. The yeast ortholog, NPR2 (accession is presented. no. X79105), shows three regions of high similarity: (1) with 32% 6441

Fig. 7. Expression analysis of NPRL2 in normal tissues and cancer cell lines. Northern analysis of expression of NPRL2 in normal tissues (A) and cancer cell lines (B). Expression in normal tissues was tested using Human Multiple Tissue Northern blot (#7765-1, Clontech). Analysis of genomic structure of NPRL2 in different cell lines using PCR primers is shown in Fig. 7C.To reduce the effect of heterogeneity, only 28 PCR cycles were used. Renal cell carcinoma cell lines: KRC/Y, HN4, Caki1, Caki2, TK10, and TK164. Small cell lung carcinoma cell lines: N417, ACC-LC5, U2020, and GLC20. A549 is a non-small cell lung carcinoma, HeLa is a cervical carcinoma, and CBMI-Ral-STO is a lymphoblastoid cell line (see Materials and Methods).

Fig. 8. Detection of homozygous deletions in the 3Ј end of NPRL2 gene in renal cell carcinoma, breast carcinoma, and ovarian carcinoma biopsies showing microsatellite instability. To reduce the effect of normal cell contamination only 28 PCR cycles were used. Double PCR was done using 6F-9R and 5F-1R PCR primer pairs (see Fig. 1). PCR for the first lane after M was done using only 5F-1R primers. (M, 100 bp markers; T, tumor biopsy; N, normal control DNA from the same organ; RCC, renal cell carcinoma) identity (53% similarity) over 152 amino acids; (2) with 36% identity bility. Inactivating homozygous deletion of the 3Ј end of NPRL2 was (50% similarity) over 119 amino acids; and (3) with 36% identity obvious in breast (357T) and ovarian (585T) carcinoma (Fig. 8). One (54% similarity) over 55 amino acids. By sequence analysis, the main might suspect that renal cell carcinoma biopsy (347T) also contains a product of NPRL2/G21 encodes a soluble protein that has a bipartite homozygous deletion. In all of the six cases, both 5F-1R and 6F-9R nuclear localization signal (residues 62-79), a protein-binding domain primer pairs produced bands with normal control DNA. Additionally, called granulin (residues 86-98), weak similarity to the MutS core we have searched public databases with the primer sequences and domain (residues 273-315), and a newly identified domain with un- didn’t find any mutations within these sequences, indicating they known function (residues 3-380), which was predicted by protein could not be a site of frequent polymorphism. To reduce the effect of families database (PFAM) and is present in many proteins. The gene normal cell contamination, we used only 28 cycles for PCR. These is highly expressed in many tissues, including lung and kidney tissues, preliminary observations are consistent with the suggestion that with alternative splicing of all of the 11 exons and introns creating a NPRL2 could participate in mismatch repair; however, more detailed variety of transcripts (altogether 31). These transcripts encode 24 studies are needed to confirm this hypothesis. different protein isoforms with different predicted cellular locations.14 The yeast ortholog, NPR2 (accession no. P39923), has yeast nitro- This information suggests that the nuclear NPRL2/G21 protein may gen permease regulator activity, which suggests that NPRL2 might be involved in mismatch repair and signaling to cell cycle checkpoints also have this activity. NPR2 is involved in posttranslational control of that activate apoptotic pathway(s). Recently, the yeast NPR2 was nitrogen permease (24). However, it could be involved in many other found to be a target of cisplatin (23), suggesting a similar function in vital processes (23, 25). The exact function of the gene in different positively responding human tumors. Cisplatin is one of the most species should be established by additional experiments. commonly applied anticancer agents and is used to treat a variety of tumors (germ-cell, advanced bladder carcinoma, adrenal cortical carcinoma, breast cancer, head and neck carcinoma, and lung carci- ACKNOWLEDGMENTS noma). It is believed to kill cancer cells by binding to DNA and We are grateful to Dr. Alena Malyukova for assistance in some experiments. interfering with its repair mechanisms, eventually leading to cell death. To test the hypothesis that NPRL2 could represent a novel 3p REFERENCES mismatch repair gene, we checked the NPRL2 gene in 2 breast, 1 renal 1. Stewart BW, Kleihues P. World Cancer Report. Lyon: IARC Press; 2003. cell carcinoma, and 3 ovarian biopsies, showing microsatellite insta- 2. Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene 2002;21:6915–35. 3. Imreh S, Klein G, Zabarovsky ER. Search for unknown tumor-antagonizing genes. 14 Web address: http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/. Genes Chromosomes Cancer 2003;38:307–21. 6442

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Jingfeng Li, Fuli Wang, Klas Haraldson, et al.

Cancer Res 2004;64:6438-6443.

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