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Identification by Functional Cloning from a Retroviral cDNA Library of cDNAs for Ribosomal Protein L36 and the 10-kDa Heat Shock Protein that Confer Cisplatin Resistance

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Received August 1, 2005; accepted January 4, 2006

	Abstract

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Based on analyses of two-dimensional gel and cDNA microarrays, our laboratory and others have demonstrated that a number of genes show altered expression during the development of cisplatin resistance (CP-r) in human cancer cells, including genes associated with DNA damage repair, proto-oncogenes, apoptosis, stress-response, and transcription factors. To verify these results and find genes that are directly responsible for CP-r, as opposed to those reflecting a secondary response induced by cisplatin treatment or resulting from CP-r, we constructed a retroviral cDNA library in the vector pLNCX2 from KB-CP.5 (KCP.5), a cell line selected in one step after exposure to cisplatin at 0.5 µg/ml. Using a library of cDNAs (1.8 x 10⁶ cDNA clones) and an intermittent cisplatin selection system to allow more effective functional cloning, 11 expressed cDNAs were identified in a primary pool of 93,000 transfected cell clones. Metallothionein 2A, a known CP-r gene, was among these 11 genes found in the transfectants after CP selection. Several other genes, including those encoding ribosomal proteins (e.g., RPL36) and heat shock protein (e.g., HSP10), were also found among the cisplatin-selected clones. Transfection of either the RPL36 cDNA or HSP10 cDNA conferred on KB-3-1 cells 2.5- to 3-fold resistance to cisplatin by clonogenic assays. A subsequent transfection also identified RPL36 as a CP-r gene. The finding that a ribosomal protein gene, RPL36, contributes to CP-r should stimulate study of the role of ribosomal proteins in multifactorial mechanisms of cisplatin resistance.

Functional cloning and retroviral cDNA libraries have been applied to define genes responsible for drug resistance, apoptosis, and so forth (Perez-Victoria et al., 2003; Mourtada-Maarabouni et al., 2004). In identifying genes related to cisplatin resistance, the approaches that have been used have a major drawback because of continuous stepwise challenge with increased cisplatin concentrations and may reflect secondary changes in genotype and phenotype during multistep selection of cisplatin-resistant cells. To explore genes primarily involved in cisplatin resistance, we inserted double-stranded cDNA into a retroviral expression vector, pLNCX2, from cisplatin-resistant KB-CP.5 cells that were selected by a single step of cisplatin at 0.5 µg/ml. An intermittent cisplatin selection procedure was designed for functional cloning, and subsequent identification of genes related to cisplatin resistance was obtained by PCR and sequencing. By this technique, 11 genes were found in the individual clones after functional cloning, including metallothionein, which has been reported to be associated with cisplatin resistance (Kelly et al., 1988), and a chaperone, 10-kDa heat shock protein (HSP10), which had been found previously to be inducible by a metal salt, cadmium chloride (Lee et al., 2002). A ribosomal protein, RPL36, was identified in this work, and in a second independent transfection, it was shown to confer 2.5-fold increased CP-r on transfected stable clones in comparison with the control vector-only-transfected cells. Therefore, this functional retroviral cloning system and modified intermittent selection provides a powerful tool for further cloning of genes able to confer CP-r.

	Materials and Methods

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Cell Lines and Cell Culture. The human epidermoid carcinoma cell lines KB-3-1 and an early-stage cisplatin-resistant cell line, KB-CP.5, which was isolated at 0.5 µg/ml cisplatin from KB-3-1 (Liang et al., 2003), were studied in this work. The RetroPack PT67 cell line was purchased from Clontech (Mountain View, CA). All cell lines were grown as monolayer cultures at 37°C in 5% CO₂ using Dulbecco's modified Eagle medium with 4.5 g/l glucose (Invitrogen, Carlsbad, CA), supplemented with L-glutamine, penicillin, streptomycin, and 10% fetal bovine serum (BioWhittaker, Walkersville, MD). Cisplatin (Sigma, St. Louis, MO) was added to the medium for KB-CP.5 (0.5 µg/ml).

Construction of a Retroviral cDNA Library. Double-stranded cDNA was synthesized from a single-step selection of the cisplatinresistant cell line KB-CP.5, which was maintained in medium containing cisplatin 0.5 µg/ml using a BD SMART cDNA library construction kit (Clontech) as described by the manufacturer. The synthesized double-stranded cDNA was digested with the restriction enzyme Sfi and then column-purified by a CHROMA SPIN-400. The lower cutoff cDNA size was 500 bp. Ligation of the Sfi-digested double-stranded cDNA to a retroviral expression vector pLNCX2 (Clontech) with Sfi-cut was performed according to the manufacturer's instructions, and the library was named KB-CP.5/pLNCX2. The retroviral cDNA library was transformed into DH5 competent cells (Invitrogen) and resulted in 1.8 x 10⁶ clones (library complexity).

Retroviral Transduction and Programmed Cisplatin Selection. Retroviral supernatants were collected from KB-CP.5/pLNCX2-transfected retroviral packaging PT67 cells and then used to infect the recipient KB-3-1 cells. After selection with G418, a pool of 96,000 clones containing the KB-CP.5 cDNA was established. A programmed selection with cisplatin is shown in detail in Fig. 1B.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Flow charts. A, strategy to functionally clone CP-r genes using a retroviral cDNA library from an early-stage cisplatin-resistant cell line, KB-CP.5 (human epidermoid carcinoma cells KB-3-1 cells selected with cisplatin 0.5 µg/ml). The flow chart shows the cloning system and is detailed in the text. B, flow diagram showing programmed/intermittent cisplatin selection for CP-r clones developed in this work, detailed in the text. C, flow chart showing isolation of 2000 neomycin-resistant clones from KB-3-1 cells, which served as a control.

Preparation of RNA, and RT-PCR. For the determination of expression levels of genes of interest, RNAs were isolated from cells using an RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RT-PCR was performed using a GeneAmp kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Specific primers for tested genes are listed below: ribosomal protein RPL36, 5'-AAATCCATTGCCCGTGTTC-3' (forward), and 5'-TCTTGGTCTTCAGGTTCTCC-3' (reverse); heat shock protein HSP10, 5'-AAGTTTCTTCCACTCTTTGACC-3' (forward) and 5'-TGAATCTCTCCACCCTTTCC-3' (reverse); -catenin, GAGAGTGTGCTGAAGATTCTG (forward), and TGATTCGTCCTTGTCACC (reverse), which were used for verification of the quality and the size of genes in the library by PCR.

Gene Transfection and Assays of Cell Resistance Levels to Cisplatin, Carboplatin, and Sodium Arsenite. Full-length cDNA for the genes encoding RPL36 and HSP10 were purchased from the American Type Culture Collection (Manassas, VA). Both genes were inserted into a mammalian expression vector, pcDNA3.1 (Invitrogen), as described by the manufacturer. Gene transfection was done with Lipofectin (Invitrogen) by following the manufacturer's instructions. Stable transfected clones were isolated after selection with G418. For testing cell sensitivities to cisplatin, carboplatin, and sodium arsenite, cells were counted after 3 days using a Coulter Counter or Cell Counting Kit (CCK8; Dojindo Labs, Gaithersburg, MD) as described in the legend to Fig. 3. A clonogenic assay was used to further confirm the ability to confer CP-r for the positive genes, such as RPL36 and HSP10. Cells were seeded into 60-mm dishes (Corning Glassworks, Corning, NY). Cisplatin at a desired concentration was introduced into each dish before cell seeding. The medium was replaced every 3 days with medium containing cisplatin at the desired concentration. Colonies were stained with methylene blue at the end of 12 days of incubation and then counted. Control cells were transfected with insert-free vector only. The values are the means of triplicate determinations.

View larger version (32K): [in this window] [in a new window]   Fig. 3. RT-PCR analysis. The specific primers for RPL36 and HSP10 are described under Materials and Methods. A, expression levels in transfectants: KB/V, KB-3-1 cells transfected with vector pcDNA3.1 only; KB/HSP10.C2, KB-3-1 cells were transfected with HSP10, clone 2; KB/RPL36.C6 and KB/RPL36.C10 cells were transfected with the RPL36 gene, two individual clones. -Actin served as control. B and C, colony-forming assay (CFA). B, killing curves measured 12 days after cisplatin selection, methylene blue-stained. KB/V, KB-3-1 cells transfected with vector pcDNA3; KB/HSP10.C2, KB cells transfected with HSP10/pcDNA3, clone 2; KB/RPL36.C6 and KB/RPL36.C10, KB cells transfected with RPL36/pcDNA3, 2 individual clones. C, the appearance of colonies in dishes of KB/V (a), KB/HSP10.C2 (b), KB/RPL36.C6, (c) and KB/RPL36.C10 (d) after exposure to cisplatin 0.3 µg/ml for 12 days as described in B. D, E, and G, cells were seeded at 5000 cells/well in a 96-well plate and were treated with the desired drug for 3 days; then cell numbers were determined by Cell Counting Kit-8 (CCK8) as described by the manufacturer. The concentrations of the drugs are indicated. F, cells were seeded at 5 x 104 cells/well in a 24-well plate and treated with cisplatin for 3 days, then counted using a Coulter Counter. The values are means of triplicate determinations.

	Results

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View larger version (66K): [in this window] [in a new window]   Fig. 2. Images of PCR products. A, the KB-CP.5/pLNCX2 library was primed by specific oligonucleotides of the retroviral vector pLNCX2 as described under Materials and Methods and then amplified by Advantage 2 PCR (CLONTECH). Two different loading amounts of KB-CP.5/pLNCX2 were applied as one third of loading (a) and full loading (b). B, -catenin was amplified by the specific primers described under Materials and Methods, showing the 2.3 kb of the full-length gene. C, images of genomic DNA PCR, showing inserts of different sizes in individual clones.

The RetroPack PT67 cell line was used to package the library. This cell line expresses a dual-tropic/polytropic envelope, and the virus produced can infect a broad range of mammalian cells. The viral titer was determined by functional assay (G418 resistance) and resulted in 570 colony-forming units/ml. Transfection of the KB-CP.5 cDNA retroviral library into PT67 cells yielded 115,000 neo-r colonies after G418 selection (Fig. 1A). The viral medium (K-VM) was collected from these cells and then infected into the target KB-3-1 cells. A pool containing 93,000 neo-r clones was obtained after G418 selection (Fig. 1A). A control pool with 2000 neo-r clones was collected (Fig. 1C) using the vector only without inserts.

An intermittent cisplatin selection was developed in this work as shown in Fig. 1B. In brief, cells were intermittently selected with cisplatin at 0.6 µg/ml for 3 days and then cultured in medium without the drug for another 3 days for recovery. A second round of 3 days of cisplatin selection followed by a recovery period of 3 days was repeated. Cisplatin-resistant colonies appeared after a period of 12 days, and much larger colonies were seen in the group of KB-3-1 cells infected with the viral medium from the KB-CP.5/pLNCX2 cDNA retroviral library (K-VM), whereas the control cells, which were infected with the viral medium from the vector without inserts, showed much smaller colonies (VM). One hundred twelve individual colonies that survived in the K-VM group were cloned and propagated in 0.4 µg/ml cisplatin for genomic DNA preparation and further PCR determination. We tried to select cells continuously with cisplatin at different concentrations, but this approach was unsuccessful because of either cells being killed at high concentrations of cisplatin or spontaneously mutated at low concentrations, generating a high background of low-level cisplatin-resistant clones similar to the control. Therefore, a recovery period during the first two rounds of cisplatin selection seems to be important for functional gene cloning, particularly under the conditions described above.

Identification of Genes in Association with Cisplatin Resistance. All 112 cisplatin-resistant clones were analyzed by genomic PCR using the primers for the retroviral vector pLNCX2 as described under Materials and Methods (i.e., only the inserts containing the vector sequences at both ends could be detected in the clones). Figure 2C shows fragment(s) of different sizes from 0.5 to 1 kb in these clones. After sequencing, 11 inserts were identified after BLAST match and are listed in Table 1. Metallothionein 2A was 1 among the 11 genes observed, and this gene had been reported to be associated with resistance to cisplatin and other heavy metals (Kelly et al., 1988; Yang et al., 1994), demonstrating that the strategy applied in this work by functional cloning and using a retroviral cDNA library has the power to identify genes related to cisplatin resistance. It should be noted that there were several ribosomal protein genes in this pool, suggesting that overexpression of the ribosomal gene family might also play an important role in protecting cells from cisplatin-induced damage.

We chose a ribosomal protein, L36 (RPL36), and a chaperone gene, HSP10, in our pool to evaluate whether these two genes have roles in CP-r. Both RPL36 and HSP10 genes were inserted into a mammalian expression vector, pcDNA3.1, respectively, and then transfected into cisplatin-sensitive (CP-s) KB-3-1 cells separately. Individual clones were isolated from the transfectants after G418 selection. Figure 3A shows that the RPL36 gene (middle) was overexpressed in two clones of RPL36-transfected cells, KB/RPL36.C6 and KB/RPL36.C10. Both of the control cells (KB/V), which were transfected with vector only, and another transfected clone KB/HSP10.C2 showed much lower levels of the gene. At the top of the figure, HSP10 was well-expressed in KB/HSP10.C2 cells, which were transfected with the HSP10 gene (approximately 2-fold higher than the control KB/V), and in two other clones isolated from the RPL36 transfection. At the bottom are shown similar expression levels of -actin in these four clones, serving as loading controls.

The sensitivities of these clones to cisplatin were then determined by a clonogenic assay. The results are shown in Fig. 3, B and C. Increased resistance to cisplatin (approximately 2.53-fold) was seen in the transfectants of RPL36, clones C6 and C10, and HSP10, clone C2, compared with the control vector-transfected only (Fig. 3C). In Fig. 3C, the amount and size of colonies in the clones expressing RPL36, clones C6 and C10 (c and d) or HSP10, clone C2 (b), were more numerous and larger than with the control vector only (a) after exposure to cisplatin 0.3 µg/ml for 12 days. These results demonstrate that, where overexpressed, the ribosomal protein RPL36 and the heat shock protein HSP10 may play roles in cisplatin resistance.

	Discussion

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A variety of approaches have recently been applied in efforts to define genes that are related to cisplatin resistance. Our previous studies demonstrated that a pleiotropic defect occurred in the cisplatin-resistant cells, including defective influx of [¹⁴C]carboplatin, and other related heavy metals or unrelated compounds, mislocalization of membrane transporters, reduced expression of folate binding protein, and increased DNA hypermethylation (Liang et al., 2003; Shen et al., 2004), indicating a multifactorial mechanism involved in CP-r.

In this work, two genes, a ribosomal protein (RPL36) and a heat shock protein (HSP10), were identified to confer cisplatin resistance by 2.5- to 3-fold using a retroviral cDNA library and functional cloning. Although the function of these two genes in CP-r is unknown, their effect may plausibly be related to increased protein synthesis and protein stabilization to protect cells from the toxic effect of cisplatin, directly or indirectly, in association with the regulation of proteins in DNA damage/repair, antiapoptotic processes, or detoxification of cisplatin. We repeated the selection using a somewhat different protocol by which cells were selected with 1 µg/ml cisplatin for 24 h, instead of intermittent exposure for a longer period of time, and then were allowed to recover with drug for a period of 8 to 10 days until cloning. By this method, we further isolated more RPL36-related genes, such as RPL36aL, found in 4 individual clones from a pool of 30 (Table 1, last entry). RPL36aL has 90% identity with the coding region of the RPL36 gene. This result provides independent evidence that the selection of the RPL36 gene family members is not a random event but reflects their association with CP-r. None of the transfected clones, however, were cross-resistant to carboplatin or sodium arsenite, as shown in Fig. 3, D and E, respectively. This does not necessarily indicate a different mechanism of resistance, because the original parent cell line, KB-CP.5, was more resistant to cisplatin (up to 50-fold), whereas the RPL36 and HSP10 transfectants showed only 2.5- to 3-fold resistance.

None of the cDNAs isolated so far from the cDNA library made from these cells confers this phenotype on the full range of resistance seen in the KB-CP.5 cells. We assume, therefore, that this phenotype either results from expression of more than one gene or that we have transfected genes whose overexpression confers CP-r by a mechanism different from that seen in KB-CP.5.

The 10-, 27-, 60-, and 70-kDa heat shock proteins and others have been linked to stress response in protein folding and unfolding in drug resistance (Mandic et al., 2002; Shan et al., 2003; Zhao et al., 2005). Ribosomal proteins have been reported recently to be associated with the regulation of apoptosis, multidrug resistance (Wendel et al., 2004), oncogenesis, and chemotherapy (Zhang and Berger, 2004). Transfection of the ribosomal protein RPL23 into gastric cancer cells was reported to induce multidrug resistance, including CP-r, and to protect cells against vinblastine-induced DNA fragmentation but had no effect on intracellular drug accumulation (Shi et al., 2004). Our finding in this work provides evidence that overexpression of RPL36 and HSP10 confers CP-r in KB-3-1 cells but does not confer resistance to sodium arsenite. The pattern of genes selected by cisplatin may reflect stress responses and cisplatin-specific resistance genes. Further functional cloning by selection with other agents, including carboplatin, would help to determine which genes are involved in general stress responses and which are unique to cisplatin.

Other genes were detected in the clones (Table 1), but RPS27, RPL41, and nucleolar protein family A, member 2, were tested individually and did not confer CP-r as shown in Fig. 3, F and G, respectively. Either these genes are not sufficient to confer CP-r but may be necessary in the context of other patterns of gene expression or they were simply false-positive results from the screen applied here where the background (vector alone) was not 0. The other genes listed in Table 1 could not be tested initially because full-length cDNAs were not readily available. Nevertheless, these results indicate that the functions of RPL36 and HSP10 in CP-r cells and their potential role in human cancers need to be further elucidated.

	Acknowledgements

 
We thank George Leiman for editorial assistance and Toru Miki for advice in preparing the cDNA library.

	Footnotes

 
This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

ABBREVIATIONS: HSP10, 10-kDa heat shock protein; RPL36, ribosomal protein L36; CP-r, cisplatin resistance; PCR, polymerase chain reaction; kb, kilobase(s); RT-PCR, reverse transcription-polymerase chain reaction.

Address correspondence to: Dr. Michael M. Gottesman, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Room 2108, Bethesda, MD 20892-4254. E-mail: mgottesman{at}nih.gov

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.017525v1 69/4/1383    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shen, D.-W. Articles by Gottesman, M. M. Search for Related Content PubMed PubMed Citation Articles by Shen, D.-W. Articles by Gottesman, M. M.