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Advances in Brief |
1 Department of Cancer Biology, Lerner Research Institute, Cleveland, Ohio; and 2 Department of Obstetrics and Gynecology, The Cleveland Clinic Foundation, Cleveland, Ohio
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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) as bait. The polß is expressed in various types of cancers. Co-immunoprecipitation–Western blot analysis confirms not only its interaction with polß but also with wild-type polß. Binding to polß indicates potential function of MGC5306 in repair pathway. Transfection of cells with MGC5306-GFP and Western blot analysis with anti-MGC5306 antibody reveal its nuclear localization. MGC5306 is expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting MGC5306 is most likely involved in carcinogenesis. An antigrowth activity and modulations of cell cycle events are identified in cells expressing siRNAMGC5306. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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protein are expressed in various types of primary carcinomas (11, 12, 13, 14, 15, 16, 17)
. Somatic mutations in exons 4, 8, 9, 11, and 12 of polß gene were identified in early stages of ovarian cancer (17)
. Most interestingly, unlike other tumors namely, colorectal, breast or RCC, a truncated 16.5-kDa polß protein is expressed in 19 of 28 ovarian tumors (67.9%). All results taken together provide evidence for a potential role of altered polß gene in tumorigenesis. Recently, Lang et al. (18)
reported that a polß variant expressed in a colorectal tumor (11)
induces a high mutation frequency and has a lower fidelity than WT-polß. Human and mouse cells expressing the polß are hypersensitive to alkylating agents (15
, 19)
. Similar to WT-polß, the polß interacts with apurinic endonuclease, poly(ADP-ribose) polymerase, and X-ray cross-complementing group1 (XRCC1) in vitro and in vivo, but it most avidly interacts with XRCC1 (20)
. More importantly, the polß bound to XRCC1 acts as a dominant-negative mutant (15
, 19
, 20)
.
In search of new protein(s) that interact with polß and thereby may play a key role in dominant-negative activity, we screened a HeLa cDNA library with polß as bait in the yeast–two-hybrid assay. In this report, we provide evidence for a positive clone, MGC5306, expression of the new gene in primary ovarian cancers and its potential role in cellular phenotype.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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in pGBKT7 (the polß construct in the DNA-binding domain). The polß in pGBKT7 was transformed into the yeast strain, AH109. The resultant transformants in AH109 was then inoculated into selection medium without tryptophan and allowed to reach an A600 nm of 0.4 to 0.6. Fifty micrograms of the HeLa cell cDNA library was sequentially transformed into AH109 containing polß following the manufacturer’s instructions. The positive transformants were identified by ß-galactosidase activity (21)
.
Tumor Specimens.
Fresh ovarian carcinomas, low malignant potential ovarian tumors, and normal ovarian tissues were acquired from the surgery unit or tissue bank, Cleveland Clinic Foundation.
PCR Amplification of MGC5306 mRNA.
RNA from human MDA468 breast, U373 glioma tumor cell lines, ovarian carcinomas, low malignant potential ovarian tumors, and normal ovary tissues were reverse transcribed and amplified (11)
with a pair of primers that would amplify the entire coding sequence of human MGC5306 (22)
. Purified PCR product was directly sequenced.
MGC5306 Construct and Cloning in pEGFP-N1 Vector.
The PCR product was cloned into the pEGFP-N1 expression vector (Clontech) at SacII site. This would ensure the right reading frame of fusion protein with tagged green fluorescent protein (GFP) at COOH-terminal of MGC5306. The size and orientation of the cloned product was assessed by restriction enzyme mapping.
Expression of MGC5306-GFP Fusion Protein in HEK293 Cells and Subcellular Localization.
HEK293 cells seeded in 35-mm plate were allowed to grow overnight. Two micrograms of the purified plasmid either pMGC5306-GFP or pEGFP-N1 vector mixed with FuGene6 transfection reagent (Roche, Indianapolis, IN) were added to the cells. The cells were incubated at 37°C for 48 hours. The fluorescent image of the transfected cells was captured by 488-nm excitation.
Fluorescent Immunohistochemistry of MGC5306 Expression and Its Colocalization with DNA Polß.
HEK293 cells seeded on chamber glass were transfected with pEGFP-polß plasmid. Cells were fixed in 2% paraformaldehyde 48 hours after transfection. Slides blotted in PBS with 0.5% BSA and 0.2% cold-water fish gelatin (PBG) were incubated with anti-MGC5306 antibody in PBG. Biotinylated secondary antibody and streptavidin conjugated with Texas Red (Molecular Probes, Eugene, OR) were applied to the slides in PBG following the manufacturer’s instructions. Vectashield mounting medium with 4',6'-diamidino-2-phenylindole (Vector Labs, Burlingame, CA) was used to mount the coverslip and to outline the nucleus. Green (polß) and red (MGC5306) images were captured by Leica Fluorescent microscope. Twenty micrograms of nuclear (15)
or cytoplasmic protein of 293 cells were separated by 12.5% SDS-PAGE and blotted with anti-MGC5306 antibody.
Coimmunoprecipitation and Western Blot Analysis.
To determine protein-protein interaction of polß and MGC5306, the pMGC5306-GFP plasmid or vector was transfected or cotransfected with pC3polß plasmid into HEK293 cells with FuGene6. Five hundred micrograms of the lysates (15)
were incubated with anti-GFP antibody overnight at 4°C. The complex was collected by secondary antibody and separated by 12% SDS-PAGE. The proteins transferred to membrane were immunodetected by Western blotting with anti-polß antibody (15
, 16) .
Peptides Synthesis and Antibody Production.
A 20-amino acid peptide of MGC5306 KAIFERFKNRKKRYKKKKKR (amino acid 78 to 97) was synthesized, HPLC purified, conjugated to carrier protein keyhole limpet hemocyanin and injected into rabbit. The rabbit antiserum was used for Western blot of tumor lysates or immunohistochemistry.
Silencing of MGC5306 Expression and Reverse Transcription-PCR Assay.
HEK293 cells (5 x 104) seeded in 6-well plate for overnight were transfected with 10, 20, 50, and 100 nmol/L of 21-bp (sense: 5'-GAAGGGAGAAAUCCUAUAUAU-3'; antisense: 5'-AUAUAGGAUUUCUCCUUCC-3'; Ambion, Austin, TX) double-stranded small interfering siRNA with siPORT Amine transfection reagent (Ambion). Specific silencing of MGC5306 expression was examined by reverse transcription-PCR analysis of MGC5306 mRNA as described previously (11
, 14)
.
Survival Assay.
The survival and viability of 293 cells were determined by total number of cells and trypan blue staining. Cells (5 x 105) seeded in triplicate in 6-well plate were transfected with double-stranded siRNAMGC5306 or untransfected. Plates were then incubated for 48 hours. Total cell number was counted. In addition, these cells were stained with trypan blue, and only live cells were scored.
Cell Cycle Analysis.
Cells transfected with SiRNAMGC5306 for 48 hours were trypsinized, washed with PBS, and fixed in 2% paraformaldehyde for 15 minutes, then in 70% ethanol overnight at –20°C. Washed cells were stained with propidium iodide (PharMingen, San Diego, CA) following the manufacturer’s instructions, and DNA content from 10,000 cells was examined by flow cytometry with a Becton Dickinson FACScan.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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was used to screen HeLa cell cDNA library for potential genes that would encode polß-interacting proteins. The pGBKT7 has a tryptophan selection marker, the cDNA library has a leucine marker, and the expression of histidine is one indicator of a protein-protein interaction in this yeast–two-hybrid system. Transformants > 2.5 x 105 were examined. The possibility of false positive colonies were eliminated by streaking the transformants from the tryptophan, leucine and histidine plates onto medium lacking these three amino acids plus adenine, an additional selection marker. Transformants with adenine expression, indicating a possible protein-protein interaction, were selected additionally for lacZ expression with ß-galactosidase. The positive colonies from the ß-galactosidase screen were PCR amplified. Some of the colonies, which contained an insert, were cloned into pCR2.1TOPO, then digested with EcoRI and sequenced. Results from one of the two identified positive clones are shown in Fig. 1A
. Colony 1 is a positive control provided in the Matchmaker Gal4 yeast–two-hybrid system kit (Clontech) shown in Fig. 1A
, Lane 1. Similarly, ß-galactosidase activity was detected in the colony with XRCC1 shown in Fig. 1A
, Lane 2. XRCC1, a known nuclear repair protein interacts with polß (20)
. All colonies, except number 4 (randomly chosen colony used as a negative control), exhibited ß-galactosidase activity, indicating that polß interacted with proteins present in the library. Colony 3 with unknown identity expressed ß-galactosidase represented in Fig. 1A
, Lane 3. The expressed sequence tag sequence of this positive clone is identical to the Homo sapiens hypothetical protein MGC (mammalian gene collection) 5306 (22)
. These results suggest that the newly identified protein, MGC5306, interacts with polß.
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, we took advantage of GFP-tagged MGC5306 and analyzed by co-immunoprecipitation–Western blot analysis. A 39-kDa polß protein was identified in the immunoprecipitated product (Fig. 1B
, bottom panel, Lane 1) when anti-GFP antibody was used to pull down the complex and Western blotting with anti-polß antibody, indicating the interaction of MGC5306 and endogenous WT-polß. In cotransfected cells, a 36-kDa polß (Fig. 1B
, Lane 2) was revealed in an immunoprecipitated product of fusion protein, suggesting binding of MGC5306 to the polß. Western blot analysis of lysates made from cells transfected with MGC5306 or cotransfected with polß by GFP antibody detected a 59-kDa fusion protein (GFP: 27 kDa; MGC5306: 32 kDa) shown in top panel, Fig. 1B
, Lanes 1 and 2, respectively. Fig. 1B
, Lane 3, shows the expression of GFP vector control. Thus, these results suggest that indeed MGC5306 interacts with polß in vivo, supporting the data revealed in yeast–two-hybrid experiment and it also binds to WT-polß.
Subcellular Localization of MGC5306.
To determine the potential expression of MGC5306, the reverse transcription-PCR product of MGC5306 mRNA from 293 cells, shown in Fig. 1C
was cloned in an EGFP-N1 expression vector at SacII site. Restriction enzyme mapping ensured the correct reading frame of fusion protein with tagged GFP. A product of 847 bp has been identified indicating expected size of the coding sequence of MGC5306. This construct was used to transfect 293 cells. Fig. 1D
shows predicted amino acids with a conserved nuclear targeting sequence. Fig. 2A
, bottom left panel, demonstrates green fluorescence in nuclei, indicating that the MGC5306 is localized in the nuclei. On the other hand, the bottom right panel shows expression of the GFP vector distributed throughout the cytoplasm and nuclei. Hence, we conclude that the fusion protein is efficiently expressed in nuclei.
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. The top right panel in Fig. 2B
shows Texas Red-stained MGC5306 in nuclei of 293 cells and green fluorescence emitted from nuclei demonstrates distinct nuclear localization of polß (Fig. 2B
, top left panel). Upon overlapping of both, the yellow stain (Fig. 2B
, bottom left panel) in the nuclei is clearly visualized, indicating both proteins reside in nuclei and in close physical distance. Fig. 2B
, bottom right panel, represents a field of cells, which were immunostained.
These findings are additionally supported by Western blot analysis that the expression of MGC5306 at the protein level is localized specifically in nuclear extract not in cytoplasm (Fig. 2C)
. Taking all results together, our observation strongly points to nuclear colocalization of MGC5306 and polß.
Expression of MGC5306 in Ovarian Carcinomas and Tumor Cell Lines but not in Normal Matched Tissues.
The interaction of MGC5306 with polß and WT-polß suggests a possible relevance of the hypothetical protein in DNA repair, especially BER, and thus in tumorigenesis. Hence, it is important to determine its expression in tumor cells. As shown in Fig. 3A
, left panel, we successfully amplified an expected 847-bp PCR product of the MGC5306 cDNA from human glioma U373 and breast carcinoma MDA468 cell lines. The expression of MGC5306 was detected in six of nine serous ovarian carcinomas but not in normal ovarian tissues represented by N1 to N3 (in all, eight normal ovarian tissues examined). MGC5306 was expressed in ovarian cancer T9 but not in the corresponding normal ovary N9. Fig. 3A
, bottom panel, shows equal loading by ß-actin expression. It seems that the MGC5306 is overexpressed in ovarian tumors in respect to normal ovarian tissues. It will be interesting to compare the MGC5306 expression levels and tumor type, stage, and metastasis status. Surprisingly, MGC5306 was not expressed in all of eight low malignant potential ovarian tumors. These results suggest that MGC5306 expression may be useful as a molecular marker for ovarian cancer and as a tool of distinguishing ovarian carcinoma from low malignant potential ovarian tumors. Thus, these data strongly indicate that the MGC5306 may be linked to ovarian tumorigenesis.
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, a 32-kDa protein, the predicted size of MGC5306 reveals in lysates of ovarian tumors represented by T4, T6, and T7. However, this protein is undetectable in normal ovarian tissue represented by N3, although ß-actin protein is expressed in this tissue. The expression of MGC5306 was detected in five of seven serous ovarian carcinomas from Western blot analysis but not in normal ovarian tissues. Hence, from the results at mRNA and protein levels, we conclude that MGC5306 is overexpressed in ovarian cancers compared with normal ovarian tissues.
To determine whether the sequence of MGC5306 cDNA (Fig. 3C)
identified in ovarian cancers matches the sequence recorded in GenBank, we examined the reverse transcription-PCR product by direct sequencing. No alteration was detected in the entire coding sequence from all samples tested. An A to G transition was observed at nucleotide 54, by which the third nucleotide of codon GAA changed to GAG (Fig. 3C)
; GAA and GAG code for glutamic acid. This silent mutation may represent nucleotide polymorphism.
siRNAMGC5306 Down-regulates Expression of MGC5306 Gene.
As shown in Fig, 4A
, Lanes 2 to 5, after 48 hours of transfection, an effective and specific silencing of MGC5306 expression was revealed with an increase of siRNA concentrations from 10 to 100 nmol/L. MGC5306 mRNA level was reduced 70% at 50 nmol/L siRNA (Fig. 4A
, Lane 4). It was additionally drastically reduced when the concentration of siRNA was increased to 100 nmol/L (Fig. 4A
, Lanes 5 and 9). Fig. 4A
, Lanes 1 and 6, show expression of MGC5306 in untransfected cells. Glyceraldehyde-3-phosphate dehydrogenase mRNA, a positive control and a negative control siRNA without homology to any known human gene sequences, showed no effect on MGC5306 mRNA expression, which are shown in Fig. 4A
, Lanes 7 and 8. These results demonstrate that siRNA targeted MGC5306 selectively and specifically silences the expression of MGC5306.
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70% of number of cell showing in Fig. 4B
.
To examine viability of cells under similar condition, we chose 100 nmol/L siRNA because this level of siRNA exhibits most impact on cells. The survival of cells was suppressed 50% (Fig. 4C)
compared with untransfected cells.
Taken together, these results provide strong evidence that siRNA-mediated silencing of MGC5306 causes an antigrowth and therefore an antisurvival effect on cells.
Effect of siRNAMGC5306 on Cell Cycle Events.
As siRNAMGC5306 causes a survival reduction, the stages in cell cycle were investigated by flow cytometry to establish a potential role of siRNAMGC5306 in cell cycle arrest or stimulation of apoptosis. Fig. 4D
shows that the S phase and apoptotic subG1 stage were markedly induced by siRNAMGC5306 in a dose-dependent manner. It is interesting to note that G2 to M/L transition stage was totally abolished when cells were transfected with 100 nmol/L siRNAMGC5306. G0-G1 stage also was substantially inhibited. These results suggest down-regulation of MGC5306 induces apoptotic cell death and arrest of cell division at S phase, indicating possible role of MGC5306 in completing DNA synthesis at this stage. However, results also indicate that probably the proliferation is stimulated.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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in vivo. MGC5306 (GenBank NM_024116) gene encoding a protein of 278 amino acids is localized on chromosome 11q21. The annotated gene consists of 10 exons spanning 11.3 kb and transcribes a 2.34-kb mRNA. There was no domain similarity with any known protein, according to National Center for Biotechnology Information and Celera databases. Expressed sequence tags from the MGC5306 region were found in cDNA libraries obtained from a variety of human cancer cell lines and fetal tissues (22)
. The MGCs or hypothetical proteins are largely based on expressed sequence tag sequences, and most of the genes have still not been characterized.
Expression of MGC5306-GFP fusion protein in nuclei and MGC5306 expression in nuclear extract strongly suggests that this novel protein resides and constitutively targeted to the nuclei of cells. A consensus bipartite motif is identified in 56% of known nuclear proteins (23)
. Analysis of MGC5306 sequence reveals a classic bipartite nuclear targeting motif within its NH2-terminal domain (amino acids 92–108; Fig. 1D
). As nuclear targeting sequences are essential for the transport of proteins into the nucleus, it is tempting to speculate that MGC5306 may have a functional role in nuclear transportation of proteins.
In addition to the bipartite nuclear targeting sequence, MGC5306 contains a number of putative phosphorylation sites, including protein kinase C and casein kinase II.
It is interesting that MGC5306 forms a protein-protein complex with polß or polß in vivo. Binding to these proteins strongly suggest that MGC5306 most likely is involved in BER because a key role of polß in this repair pathway has been established. It is also important to evaluate whether MGC5306 has a role in the dominant-negative activity of polß, especially in hypersensitivity of cells expressing the polß protein in response to DNA-alkylating agents, leading to carcinogenesis. The immediate future goal is to understand functions of MGC5306 in DNA repair when it interacts with polß.
With the completion of the human genome project, more attention is being focused on how those sequence data be interpreted into protein structure, function, and role in cellular growth and proliferation. Our data provide the first evidence for the expression of the novel hypothetical protein MGC5306 localized in the predicted nuclei of cells. More importantly, its expression in primary ovarian carcinomas but not in normal corresponding tissues, and low malignant potential ovarian tumors suggest potentially MGC5306 is involved in ovarian tumorigenesis. Clinically, low malignant potential ovarian tumors are considered as being between benign and malignant tumor, with a higher level of epithelial proliferation and atypia (24) . Thus, these results indicate that MGC5306 perhaps can be used as a potential molecular marker for ovarian cancers and may also help to distinguish ovarian carcinomas from low malignant potential ovarian tumors. Study of the potential differential expression of MGC5306 in other types of carcinomas and its relevance to tumorigenesis is under way.
Cellular phenotype expressed as growth and survival was specifically suppressed when expression of MGC5306 was down-regulated by siRNAMGC5306, indicating clearly that MGC5306 has a growth advantage to cells. More importantly, siRNAMGC5306 selectively induces apoptosis and arrests proliferating S phase, suggesting that this novel gene may play a significant role in maintenance of normal cell cycle events. Additional studies should shed light on the precise mechanisms underlying the role of MGC5306 in cell cycle regulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Sipra Banerjee, Department of Cancer Biology/NB40, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 444-0631; Fax: (216) 445-6269; E-mail: banerjs{at}ccf.org
Received 8/ 4/04. Revised 9/ 2/04. Accepted 9/13/04.
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  L. Wang, N. Bhattacharyya, T. Rabi, L. Wang, and S. Banerjee Mammary carcinogenesis in transgenic mice expressing a dominant-negative mutant of DNA polymerase {beta} in their mammary glands Carcinogenesis, June 1, 2007; 28(6): 1356 - 1363. [Abstract] [Full Text] [PDF]
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