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Tumor Biology |
Breast Cancer Program, Karmanos Cancer Institute [M. P. V. S., A. L., D. W. V., N. K.], Department of Pathology [M. P. V. S., D. W. V., H. H.], and Center for Molecular Medicine and Genetics [H. H.], Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin on centrosomes. This physical association of Rad6 with centrosomes is maintained throughout the interphase and mitotic phases of the cell cycle. The Rad6 protein exhibits notable alterations in distribution during interphase and mitotic stages of the cell cycle that are compatible with its function as a transcription factor. These findings suggest that Rad6 is an important ubiquitin-conjugating enzyme that may play a significant role in the maintenance of genomic integrity of mammalian cells and that an imbalance in the levels and activity of Rad6 could lead to chromosomal instability and transformation in vitro. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Rad6 group is concerned with postreplication or "error-prone" repair (2) . The Rad6 gene encodes a Mr 17,000 protein (3) , which is one of a group of ubiquitin-conjugating (E2) enzymes (4) that covalently add ubiquitin to selected lysine residues. The Rad6 pathway appears to be regulated by post-translational modification of target proteins with ubiquitin, which commits them to rapid proteolysis. The Rad6 gene of Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair, induced mutagenesis, and sporulation (5 , 6) . rad6 mutant phenotypic effects include slow growth, severe defects in induced mutagenesis, extreme sensitivity to UV, X-ray, and chemical mutagens, and hypersensitivity to antifolate drug metabolites (7) . The diversity of the phenotypes of rad6 mutants suggests that the Rad6 gene product is central to cell cycle regulation. All of the functions performed by the Rad6 protein appear to result from ubiquitination, because replacement of the conserved Cys 88 with serine produces a totally null phenotype. Although all of the E2s characterized to date are structurally related, they fall into several functionally distinct categories. The yeast CDC34 (UBC3) E2 is required for G1 to S phase transition of the cell cycle (8) , whereas the yeast Rad6/UBC2/E2 is involved in a variety of processes including DNA repair, mutagenesis, and cell proliferation (6) .
Rad6 is highly conserved among eukaryotes. Two closely related human DNA repair genes, HHR6A and HHR6B (human homologues of yeast Rad6), encode ubiquitin-conjugating enzymes (E2), and complement the DNA repair and UV mutagenesis defects of the S. cerevisiae rad6 mutant (9) . HHR6A and HHR6B share 95% identical amino acid residues and are localized on human chromosome Xq24-q25 and 5q23-q31, respectively (10) . Inactivation of the gene encoding the mammalian homologue of yeast Rad6, HR6B, in mice leads to male sterility (11) . Experiments described here show for the first time that HR6B is overexpressed in mouse and human breast cancer lines and tumors, and that constitutive overexpression of HR6B induces formation of multinucleated cells, centrosome amplification, abnormal mitosis, aneuploidy, and transformation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human Breast Epithelial Cell Lines.
MCF10A, MCF10AT, MCF10ADCIS.com, MCF-7, and MDA-MB-231 cells were also used. MCF10A cells are normal-behaving human breast epithelial cells that lack tumorigenicity in nude mice and are unable to support anchorage-independent growth (14)
, whereas MCF10AT and MCF10ADCIS.com are T24-Ha-ras-transformed MCF10A cells that produce preneoplastic (15)
or DCIS3
(16)
lesions, respectively, when implanted in immunodeficient mice. MCF-7 (tumorigenic) and MDA-MB-231 (metastatic) human breast cancer cells were obtained from Cell Resource Core of Karmanos Cancer Institute or purchased from American Type Culture Collection (Manassas, Virginia), respectively.
Cell Culture.
Mouse mammary tumor sublines were grown in DMEM supplemented with 5% FCS, 5% newborn calf serum, 1 mM nonessential amino acids, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. MCF10A and MCF10AT cells were maintained in DMEM/F-12 medium supplemented with 2.5% horse serum, 0.02 µg/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, 10 µg/ml of insulin, 0.1 µg/ml cholera toxin, 100 units/ml penicillin, and 100 µg/ml streptomycin. MCF10ADCIS.com and MDA-MB-231 cells were grown in DMEM supplemented with 10% FCS, and MCF-7 cells were maintained in DMEM/F-12 supplemented with 5% FCS and 10 µg/ml insulin.
mRNA Differential Display.
Total cellular RNA was isolated using the TRIzol reagent kit (Life Technologies, Inc., Grand Island, NY) following the manufacturer’s protocol. Before mRNA differential display, DNase I treatment was performed on the RNA samples using the Message Clean kit (GenHunter Corp., Nashville, TN) and differential display of cDNA fragments was performed essentially as described by Liang et al. (17)
. The radioactive PCR products were electrophoresed on 6% acrylamide-8 M urea gels in Tris-borate EDTA buffer (pH 8.0) at 35 W for 3 h, and the gel was dried and subjected to autoradiography. Selected bands were identified, and the corresponding slices on the dried gels were excised and eluted by incubation in 50 µl of 10 mM Tris-HCl-1 mM EDTA buffer (pH 8.0) at 60°C for 1 h. Gel pieces containing differentially displayed bands of interest were reamplified by PCR, subcloned into pCR-telomeric repeat amplification protocol vector and sequenced using primers provided in the pCR-telomeric repeat amplification protocol cloning system (GenHunter Corp.). Gas1 (18)
and the HR6B/E2B/Rad6 cDNAs were isolated using 5'-AGGTGACCGT-3' (AP3) and T11C primers for PCR amplification (GenHunter Corp.).
Full-length HR6B cDNAs were amplified by RT-PCR from RNAs of 168FAR, 66cl4, and 4T1 tumor sublines, and normal BALB/c mouse liver using the forward and reverse primers 5'-AGCTGCGGAGCATGTCG-3' (+17/+33) and 5'-AAGGATGAGCAGACCAGG-3' (+574/+553; Accession no. NM_009458), respectively. PCR amplification was performed for 25 cycles at 95°C for 1 min, 52°C for 2 min, and 72°C for 3 min. The PCR products were additionally characterized by Southern blot analysis using: (a) the 240-bp cDNA fragment isolated from differential display; and (b) a full-length HHR6B cDNA prepared from the normal human breast epithelial cell line MCF10A using primers +330/+348 and +935/+914 (Accession no. M74525). The amplified cDNAs were sequenced by Cyclist DNA sequencing kit (Stratagene, La Jolla, CA) and sequence data subjected to similarity search at nucleotide and amino acid levels using the GenBank databases.
RT-PCR Analysis of HR6B/E2B/Rad6 Expression.
Total RNAs (2 µg) from 67, 168FAR, 4T07, 4T1, and 66cl4 tumor sublines were digested with RNase-free DNase I and reverse transcribed using random hexamers and Superscript II (Life Technologies, Inc., Rockville, MD). Relative levels of HR6B mRNA expression were determined by RT-PCR using primers +17/+33 and +114/+97 (Accession no. NM_009458) as forward and reverse primers, respectively, and conditions that yielded a detectable PCR product using the minimum number of amplification cycles. In addition to the primers for the HR6B gene, a set of primers designed to recognize GAPDH cDNA was used in each reaction as an internal control for the amount of cDNA tested. The GAPDH-specific primers were forward, 5'-CATTGACCTCAACTACATGGT-3' (+186/+206) and reverse, 5'-GGATCTCGCTCCTGGAAGA-3' (+320/+302; Accession no. XM_006959). The PCR products were separated by agarose gel electrophoresis, transferred, and blots probed with the full-length HR6B (PCR amplified using primers +17/+33 and +509/+493; Accession no. NM_009458) and GAPDH cDNAs as the probes. The relative intensities of the HR6B and GAPDH hybridizable bands were quantitated by densitometry (Model 300A densitometer; Molecular Dynamics, Sunnyvale, CA).
Preparation of Rad6 Antibody and Western Blotting.
Antibody to Rad6 was generated by multiple immunization of New Zealand White rabbits with a synthetic peptide (K plus amino acid residues 131–152; Accession no. NM_009458) that is conserved 100% in mouse and human HR6B, and 91% in human HR6A. For Western blotting, cell lysates from exponentially growing mouse and human breast cells were prepared in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of leupeptin, pepstatin, antipain, and 1 mM sodium orthovanadate, and proteins (50 µg) from each lysate were separated by SDS-PAGE and transblotted onto Immobilon P membranes. The blots were stained with anti-Rad6 antibody. After detection, blots were stripped and reprobed with a rabbit polyclonal Gas1 protein antibody, a kind gift from Dr. G. Del Sal (International Center for Genetic Engineering and Biotechnology, Consortium for Interuniversity, Biotechnology Laboratories, Trieste, Italy). Loading of protein was monitored by reprobing stripped membranes with mouse anti-ß-actin antibody. Rad6 (and Gas1) and ß-actin protein bands were visualized with antirabbit or antimouse IgG coupled to HRP, respectively, using enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). The relative amounts of Rad6 (HR6A/HR6B) protein(s) to ß-actin bands were quantitated with a scanner-densitometer (Molecular Dynamics).
Immunofluorescence Microscopy.
For immunofluorescence staining, mouse mammary tumor sublines 67, 168FAR, 66cl4, and 4T1, and MCF10A human breast epithelial cells were grown on coverslips and fixed in methanol:acetone (1:1, v/v) at -20°C. Cells were preincubated with 2% horse serum/PBS and incubated with anti-Rad6 antibody. To assess centrosome number or colocalization of Rad6 with centrosomes, vector-transfected and Rad6-overexpressing MCF10A clones fixed in methanol-acetone were stained with mouse monoclonal antibody to -tubulin (Zymed Labs, San Francisco, CA), or a 1:1 mixture of anti-Rad6 antibody plus -tubulin antibody. Rad6 and -tubulin were detected with FITC-conjugated goat antirabbit IgG and Texas Red-conjugated goat antimouse IgG, respectively, and scored for the number of centrosomes. Nonbinding mouse or rabbit IgG was used as a control in all of the double-labeling experiments. Slides were counterstained with 4',6-diamidino-2-phenylindole (Molecular Probes), and the number of abnormal mitoses was determined at x100 magnification and expressed as a percentage of the total number of mitotic nuclei per 300 nuclei scored. Representative cells at different stages during mitosis were also scored to evaluate Rad6 expression and localization. All of the images were collected on a Olympus BX-4 fluorescence microscope equipped with Sony high resolution/sensitivity CCD video camera.
Immunohistochemistry.
Cryostat or formalin-fixed, paraffin-embedded human breast carcinoma tissue sections were incubated with anti-Rad6 antibody followed by biotinylated antirabbit IgG secondary antibody and HRP-conjugated streptavidin. Nuclei were counterstained with hematoxylin. Control sections were stained with secondary antibody only.
Rad6-associated Histone H2B Ubiquitination Activity.
Cell extracts of mouse tumor sublines 67, 168FAR, 4T07, 66cl4, and 4T1 containing equivalent amounts of total protein (100 µg) were immunoprecipitated with anti-Rad6 antibody or with an equivalent amount of normal rabbit IgG. Immune complexes were pelleted after incubation with protein A/G-Sepharose, washed in lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride], and the washed pellets rinsed with reaction buffer [50 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 2 mM ATP, 0.2 mM DTT, 100 µg/ml BSA]. The rinsed pellets were resuspended in the same buffer supplemented with 0.5 µg Histone H2B (Sigma Chemical Co., St. Louis, MO), 0.5 µg ubiquitin (Sigma Chemical Co.), and 0.2 µg ubiquitin activating enzyme, E1 (Boston Biochem, Boston, MA) and incubated for 30 min at 30°C. Some reactions were performed with the same assay buffer but lacking ATP. The reaction mixtures were run on reducing 17% polyacrylamide gels, electroblotted, and subjected to Western analysis with mouse antiubiquitin antibody (Zymed Labs). Ubiquitinated histone H2B and free ubiquitin were visualized with antimouse IgG coupled to HRP using an enhanced chemiluminescence kit (Amersham Corp.).
Generation of Stable HHR6B Transfectants and Analysis.
Full-length wild-type HHR6B cDNA was subcloned into the BamHI site of the mammalian expression vector pCMVneo (Promega Corp., Madison, WI) for high-level expression under the transcriptional control of the cytomegalovirus promoter. Circular plasmid DNA (either empty vector or sample construct) was transfected into MCF10A cells by Mirus Trans IT-I transfection reagent (PanVera Corp., Madison, WI). Stable transfectants were selected by resistance to G418 selection (500 µg/ml) for 3 weeks. Selected resistant clones were isolated, expanded, and maintained in the presence of G418. Expression of the transfected gene was monitored by RT-PCR with primers specific for the vector and the exogenous gene on DNase-treated RNA samples, Northern, and Western blot analysis.
Growth in Soft Agar.
Vector-transfected or HHR6B-overexpressing MCF10A clones (2 x 104 cells) were seeded in 2 ml of 0.33% agar in DMEM-F12-supplemented medium as described above for propagation of MCF10A cells. This suspension was layered over 1 ml of 0.9% agar medium base layer and dishes incubated at 37°C in 5% CO2/95% O2 for 4 weeks with twice weekly medium changes. All of the cultures were examined 24 h after plating, and cell aggregates that might bias final results were marked. Plates with >10 aggregates were discarded. CFE was calculated by dividing the number of colonies >50 µm (sized using a calibrated ocular grid) by the number of cells seeded. Ten microscopic fields were counted to calculate the total number of colonies/well from the whole well (19)
. Reported values are the average count from triplicate wells. The number of colonies in different size ranges (50–100 µm and >100 µm) was calculated in the same manner.
SKY.
The probe mixture containing 24 differentially labeled, chromosome-specific painting probes and Cot-1-blocking DNA (SKY kit; Applied Spectral Imaging, Carlsbad, CA) was denatured and hybridized to denatured metaphase chromosomes (20
, 21)
according to the protocol recommended by the manufacturer. After hybridization and washing, the chromosomes were counterstained with DAPI. Image acquisitions were performed using a SD200 Spectracube system mounted on a Zeiss Axioskop microscope with a custom designed optical filter (SKY-1; Chroma Technology, Brattleboro, VT). The emission spectra were then converted to the pseudocolors matching the fluorchrome combinations of each chromosome. For parental MCF10A and each clone, at least 9–10 metaphases were analyzed.
Statistical Analysis.
Specific differences between parental vector-transfected MCF10A cells and HHR6B-overexpressing MCF10A cells were examined by Student’s t test for centrosome numbers, or number of cells containing multiple/giant nuclei or showing abnormal mitosis. Statistical significance was determined using Student’s t test with P < 0.005 considered as statistically significant.
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RESULTS |
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. A BLAST search for sequence homologues in the GenBank database revealed that the abundantly expressed 240-bp cDNA is the mouse homologue of the yeast ubiquitin-conjugating enzyme Rad6 and matched 93% and 100% with the human HR6B (Accession no. M74525) and mouse E2B (Accession no. NM_009458) genes, respectively, whereas the down-regulated 290-bp cDNA matched 100% with the mouse gene Gas1 (18)
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are in agreement with those from differential display (Fig. 1a)
. HR6B mRNA levels in 4T07 and 4T1 cells were 
35-fold higher than line 67, and approximately 15–20-fold, respectively, higher than lines 168FAR and 66cl4 (Fig. 1b)
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Cell lysates prepared from mouse mammary tumor sublines (67, 168FAR, 4T07, 66cl4, and 4T1) or human breast cells MCF10A (normal), MCF10AT (preneoplastic), MCF10ADCIS.com (DCIS lesions), MCF-7 (tumorigenic), and MDA-MB-231 (metastatic) were subjected to SDS-PAGE and Western blot analysis with rabbit anti-Rad6 antibody. As expected for the mouse HR6B/E2B/Rad6 protein, the antibody recognized a Mr 17,000 protein from all of the cell extracts. Levels of the Rad6 protein detected in the lysates of mouse tumor sublines are in agreement with those observed by RT-PCR (Fig. 1b)
. The 66cl4, 4T07, and 4T1 cells expressed 10-, 15-, and 25-fold higher steady-state levels of Rad6 protein, respectively, when compared with lines 67 and 168FAR (Fig. 1c)
. The human HR6A and HR6B proteins share 95% identical amino acid residues. Because the synthetic peptide used for generation of the HR6B antibody differs from HR6A by only two amino acid residues, levels of Rad6 protein expressed by HR6A and B forms are not distinguishable. Thus, the Mr 17,000 immunoreactive protein(s) detected in the human cells are referred to as Rad6 rather than HR6A or B. Western blot analysis of human breast cells revealed approximately 3–4-fold higher levels of Rad6 protein in MCF10ADCIS.com, MCF-7, and MDA-MB-231 breast cancer cells as compared with MCF10A and MCF10AT cells after normalizing for loading with ß-actin antibody (Fig. 1d)
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Overexpressed HR6B mRNA Is Wild Type.
HR6B cDNAs were amplified by RT-PCR from 67 (low Rad6 expressor), 4T1 (Rad6 overexpressor), and normal BALB/c mouse liver using primers designed to yield full-length HR6B cDNAs as described in "Materials and Methods." The amplified cDNAs were subjected to direct sequence analysis, and sequences of 67 and 4T1 Rad6 cDNAs compared with those from the normal mouse liver. No alterations were detected (data not shown), and the sequence matched 100% to that reported for the mouse HR6B/E2B/Rad6 mRNA indicating that the Rad6 overexpressed in the highly metastatic subline 4T1 is wild type.
Immunocytochemical Localization of Rad6 Protein in Mammary Tumor Cells and Human Breast Carcinomas.
Results of immunofluorescence microscopy not only confirmed the abundant presence of Rad6 protein in metastatic 4T1 cells (Fig. 1c)
but also showed a dramatic difference in cellular distribution of Rad6 between normal, nonmetastatic and metastatic breast epithelial cells. Whereas MCF10A and 168FAR cells show predominant localization of Rad6 in the cytoplasm with diffuse or punctate staining in the nucleus, Rad6 protein is predominantly localized as large aggregates in the nuclei of metastatic 4T1 cells (Fig. 2a)
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. Low levels of Rad6, predominantly localized in the lumen, were observed in the ducts of normal breast (Fig. 2b
, panel A), and in ducts of normal mammary epithelium adjacent to tumor (Fig. 2b
, panel E). Breast tissues with adenosis revealed moderate Rad6 immunoreactivity Fig. 2b
, panel B). In contrast to weak Rad6 staining in normal breast epithelium, breast tumors with DCIS (Fig. 2b
, panels C and D) or invasive cancer (Fig. 2b
, panels E and F) exhibited intense Rad6 reactivity. Whereas Rad6 immunostaining is localized in the cytoplasm of cells from normal or benign breast tissues, it is interesting to note that a significant proportion of DCIS and invasive breast carcinomas exhibited nuclear immunoreactivity. Among focally to strongly immunoreactive tumors, tumor cell staining was cytoplasmic in 5 (2, stage I; 3, stage II), nuclear in 2 (stage III), and combined nuclear and cytoplasmic in 4 (1, stage 1; 1, stage II; and 2, stage III).
Ubiquitin-conjugation Activity of Rad6 Protein in Tumor Cells.
To determine whether the mouse tumor sublines of variant metastatic capacities and Rad6 expressions differed in their Rad6-mediated ubiquitin- conjugating activity, we tested the ability of Rad6 immunoprecipitated from cell lysates to ubiquitinate histone H2b. Results of Fig. 3
demonstrate both the validity of the anti-Rad6 antibody and the presence of functionally active Rad6 in the extracts of tumor sublines as immunoprecipitable Rad6 from all of the tumor sublines, regardless of their metastatic capacity, had the ability to conjugate one or two molecules of ubiquitin to histone H2b. However, contrary to expected results, metastatic sublines exhibited lower histone ubiquitin-conjugating activity as compared with nonmetastatic sublines that was not proportional to levels of endogenous Rad6. The histone ubiquitin-conjugating activity is dependent on Rad6 enzymatic activity, because assays performed in the absence of ATP or extracts immunoprecipitated with the corresponding normal IgG fail to conjugate ubiquitin to histone.
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. Clones 1 and 5 overexpressing Rad6 mRNA (50-fold higher than vector-transfected parental cells) and protein displayed an increase in the number of cells exhibiting nuclear pleiomorphism, multinucleated phenotypes, and supernumerary nucleoli (Fig. 4b)
. The generations of multinucleated cells are probably the result of both abnormal mitosis and cell-cell fusion. Evidence for the latter event is deduced from the observation that adjacent nuclei of two or more Rad6-overexpressing cells are found frequently to polarize toward one another apparently promoting cell-cell fusion with the cell membranes separating them often becoming less prominent (Fig. 4b)
. It is interesting to note that whereas parental MCF10A cells showed no cells with polarized nuclei on day 4 of plating, Rad6-overexpressing clones 1 or 5 frequently displayed cells with two or more polarized nuclei that widely ranged from 5 to 25%, which is probably reflective of dynamic cell behavior. Quantitative analysis of multinucleated cells revealed that in contrast to parental MCF10A cells that had <0.4% of cells containing multiple or giant nuclei, both Rad6-overexpressing clones 1 (14.5 ± 4; P < 0.005) and 5 (25.2 ± 8.9; P < 0.001) exhibited a significant increase in the percentage of cells containing multiple or giant nuclei. Although a proportion of these cells undergo senescence, the majority retained their proliferative capacity as observed by expression of PCNA (Fig. 4c)
. Colocalization of Rad6 with PCNA (Fig. 4c)
was observed in some cells implicating the intimate association that Rad6, a DNA repair protein, might have with DNA replication factors during the S phase of the cell cycle.
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-tubulin antibodies in mitotic and nonmitotic nuclei revealed the presence of supernumerary centrosomes in constitutively HHR6B-overexpressing MCF10A clones 1 and 5 as compared with cells transfected with the empty vector. Whereas 98% of parental MCF10A cells contained two or three centrosomes per cell, 25% of HHR6B-overexpressing clones displayed more than four centrosomes per cell including a significant proportion with >10 centrosomes in clone 5 (Fig. 5, a and b)
. These cells contained either a single giant nucleus or multiple multilobed nuclei. Double immunofluorescence labeling and image-merging experiments demonstrated colocalization of Rad6 with centrosomes in interphase cells and at each spindle pole in mitosis (Fig. 5c)
. Centrosome amplification observed in HHR6B-overexpressing clones was associated with a 4–8 fold increase in the number of multipolar mitosis (more than three and up to six spindle poles) as compared with MCF10A cells transfected with control vector (Fig. 6, a and b)
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, panel A) or focally concentrated in the nucleolus during S and/or G2-phases (Fig. 7
, panel B). On entry into mitosis, i.e., after the breakdown of the nuclear membrane, Rad6 diffuses throughout the cytoplasm, and the pattern of fluorescence corresponds to the shape of the cell. However, the fluorescence from Rad6 is clearly reduced in correspondence to the volume occupied by the condensed chromosomes indicating that Rad6 is not associated with DNA during mitosis (Fig. 7
, panels C–F). After cell division and reformation of nuclear membranes (Fig. 7
, panels G and H), Rad6 protein is redistributed throughout the nucleus suggesting that the protein is concentrated in the nuclei by passage through the nuclear membrane.
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correlated with chromosomal rearrangements, HHR6B-overexpressing clones 1 and 5, and the parental vector-transfected MCF10A cells were analyzed separately by SKY. Although clones 1 and 5 retained the translocation markers t(5;9), t(19;6), and t(3;17), and trisomy 20 observed in parental cells, it is interesting to note that both the HHR6B-overexpressing clones exhibited a tendency to lose chromosomes, because their chromosomal numbers ranged from 42–48 for clone 1 and 31–48 for clone 5 as compared with 46–48 for vector-transfected MCF10A cells (Table 1)
. Several new aberrations not observed in parental cells were also found. These included translocations 6;9, 16;1 in 100% of clone 1 and trisomy 1 in 40% of mitotic clone 5 cells.
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10% with 50% of the colonies ranging 50–100 µm in diameter. HHR6B-overexpressing clone 5 had a CFE of 40% with 60% of the colonies ranging from 50–100 µm and a third of them with diameter >100 µm. Interestingly, whereas a majority of the colonies formed by clone 5 cells remained viable and proliferative, colonies formed by clone 1 exhibited an increase in tendency to undergo apoptosis. These data suggest that constitutive Rad6 overexpression confers the ability for anchorage independent growth; however, continued survival and proliferation of the colonies are dependent on additional chromosomal alterations incurred by cells. |
DISCUSSION |
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Because the antibody used in our studies probably fails to distinguish between HR6A and B forms of Rad6, it is not clear whether the elevated levels of Rad6 protein observed in metastatic cells are derived from either one or both forms of Rad6 genes. It is also not clear whether the high levels of Rad6 protein and its predominant (aberrant?) localization in the nuclear subcompartment is indicative of its role as a direct contributor of genomic instability and, hence, progression, or whether the presence of elevated levels of Rad6 simply reflect the increased mutation rates in malignant tumors. Interestingly, Rad6 protein exhibits notable alterations in its distribution in the interphase and mitotic nuclei that is compatible with its function as a transcription factor. Stable constitutive overexpression of HHR6B in near diploid normal-behaving MCF10A cells resulted in generation of multinucleated cells, abnormal centrosome numbers, multipolar mitosis, and transformation in vitro. Although the mechanism(s) responsible for generation of multinucleated cells (i.e., abnormal mitosis and/or cell-cell fusion) are yet to be determined, one possibility is that high levels of Rad6 may mediate increased ubiquitin-mediated degradation of proteins on cell membrane.
Whereas a small subset of cancers exhibit genetic instability primarily at the nucleotide level, most breast cancers exhibit instability at the chromosomal level resulting in losses and gains of whole chromosomes, or large portions thereof (22
, 23) . That Rad6 overexpression results in aneuploidy is confirmed by SKY analysis, because HHR6B-overexpressing clones 1 and 5 were found to have fewer chromosomes ranging from 42–48 or 31–48, respectively, than the parental vector-transfected MCF10A cells that had 46–48 chromosomes. Generation of aneuploidy appears to be an initiation step in these cells, because the parental MCF10A cells are near-diploid and lack the ability to support anchorage-independent growth in soft agar (14)
. In contrast, Rad6-overexpressing MCF10A cells exhibit a deviation from the normal number of chromosomes, and show centrosome amplification, multipolar mitosis, and the ability to grow in soft agar. Because the clones analyzed overexpress up to 50-fold greater levels of Rad6 than vector-transfected parental cells and 10-fold greater levels than breast cancer cell lines, it will be interesting to verify whether cells expressing ectopic Rad6 at levels observed in breast cancer cell lines exhibit aneuploidy and whether these effects can be negated by overexpression of mutant rad6.
Aneuploidy is the predominant class of genomic instability found in breast, colorectal, prostate, and other solid cancers in general (24, 25, 26, 27) . Given the relationship of centrosome function to cell polarity and to maintenance of genomic integrity, understanding the mechanisms that lead to aberrant centrosomes, their interaction with specific proteins, and the degree and nature of centrosomal defects may have predictive value in regard to patient prognosis. Centrosome amplification or dysregulation in centrosome duplication results in assembly of aberrant mitotic spindles that result in misegregation of chromosomes and aneuploidy (28) . That Rad6 may play an important role in the maintenance of genomic integrity is strengthened by the observation that it is associated with centrosomes throughout the interphase and mitotic phases of the cell cycle, and displays striking changes in its distribution during different stages of the cell cycle. A noteworthy consequence of Rad6 overexpression is abnormal mitosis. It is conceivable that the loss of chromosome(s) evidenced by SKY analysis resulted from multidirectional forces impacted on a single chromosome in a multipolar spindle (29) . Consequently, the daughter cells would receive abnormal numbers of chromosomes and become aneuploid as supported by our SKY data. These data show that HHR6B overexpression can cause aberrant chromosomal partitioning at mitosis culminating in a catastrophic loss or gain of chromosomes that result in either cell death or survival through malignant transformation.
Centrosomes are abnormal in number, form, and function in a number of human tumors although the mechanisms by which centrosomal anomalies arise is unknown. Phosphorylation of centrosomal proteins in Drosophila (29) and vertebrates (30 , 31) have been reported to influence microtubule nucleation and dynamics at the centrosomes (32) . In mammalian cells, cdc2, NIMA, and PLK1 kinases have been implicated in centrosome duplication, maturation, and separation (33, 34, 35, 36) . Because Rad6 protein is associated with centrosomes throughout the interphase and mitotic phases of the cell cycle, Rad6 protein may play an important regulatory role via regulated ubiquitination and proteolytic degradation of centrosomal proteins. Thus, an imbalance in the levels of Rad6 protein in the cell or that associated with centrosomes could lead to defects in centrosome duplication, maturation, and function, which could induce aneuploidy. Taken together, these data suggest that centrosomes may provide a platform for assembly and functioning of several activities, and that alteration or dysregulation in the activities of specific molecules could directly impact centrosome function as part of tumorigenic process. Identification of natural substrates of Rad6 on centrosomes will help in understanding its role in maintenance of centrosome structure and function, the disruption of which could result in anomalous centrosome amplification and chromosome segregation in tumor cells. These may, in turn, provide new insights in development of new drugs for therapy of tumors with chromosome instability.
In summary, our findings suggest that Rad6 is an important ubiquitin-conjugating enzyme that may play a significant role in the maintenance of genomic integrity of mammalian cells and that an imbalance in the levels and activity of Rad6 could lead to chromosomal instability and transformation in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by United States Army Medical Research and Materiel Command Grant DAMD17-99-I-9443 (to M. P. V. S.); and NIH Grant CA22453 (Cancer Center Support Grant to the Karmanos Cancer Institute). 
2 To whom requests for reprints should be addressed, at Breast Cancer Program, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201. Phone: (313) 833-0715, extensions 2326/2259; Fax: (313) 831-7518; E-mail: shekharm{at}karmanos.org
3 The abbreviations used are: DCIS, ductal carcinoma in situ; Gas, growth arrest-specific; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; CFE, colony-forming efficiency; PCNA, proliferating cell nuclear antigen.
Received 6/29/01. Accepted 1/28/02.
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 S. Masuyama, S. Tateishi, K. Yomogida, Y. Nishimune, K. Suzuki, Y. Sakuraba, H. Inoue, M. Ogawa, and M. Yamaizumi Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions Genes Cells, August 1, 2005; 10(8): 753 - 762. [Abstract] [Full Text] [PDF]
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 S. Miyase, S. Tateishi, K. Watanabe, K. Tomita, K. Suzuki, H. Inoue, and M. Yamaizumi Differential Regulation of Rad18 through Rad6-dependent Mono- and Polyubiquitination J. Biol. Chem., January 7, 2005; 280(1): 515 - 524. [Abstract] [Full Text] [PDF]
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Y. Okamoto, T. Ozaki, K. Miyazaki, M. Aoyama, M. Miyazaki, and A. Nakagawara UbcH10 Is the Cancer-related E2 Ubiquitin-conjugating Enzyme Cancer Res., July 15, 2003; 63(14): 4167 - 4173. [Abstract] [Full Text] [PDF]
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 A. Lyakhovich and M. P. V. Shekhar Supramolecular Complex Formation between Rad6 and Proteins of the p53 Pathway during DNA Damage-Induced Response Mol. Cell. Biol., April 1, 2003; 23(7): 2463 - 2475. [Abstract] [Full Text] [PDF]
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R. Bahar, J. O-Wang, K. Kawamura, M. Seimiya, Y. Wang, M. Hatano, S. Okada, T. Tokuhisa, T. Watanabe, and M. Tagawa Growth Retardation, Polyploidy, and Multinucleation Induced by Clast3, a Novel Cell Cycle-regulated Protein J. Biol. Chem., October 11, 2002; 277(42): 40012 - 40019. [Abstract] [Full Text] [PDF]
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); breast tissues with adenosis exhibit moderate Rad6 immunoreactivity (B); and breast tumors with DCIS (C and D) or invasive cancer (
E and F) exhibit intense Rad6 staining. D and F, higher magnifications of a DCIS and malignant breast tumor, respectively. Whereas Rad6 immunostaining is localized in the cytoplasm of cells from normal or benign breast tissues, note the presence of distinct patterns of Rad6 distribution in a DCIS: cytoplasmic staining (long black arrow) in the peripheral cells versus nuclear staining (
) in the core (D). A, B, C, and E, bars, 10 µm; D and F, bar, 4 µm.
). C, confluent morphologies of confluent HHR6B-overexpressing MCF10A cells. Bars, 1 µm. c, immunofluorescence analysis of Rad6 and PCNA expression in HHR6B-overexpressing MCF10A clone 5 and parental MCF10A cells. HHR6B-overexpressing MCF10A clones exhibit intense Rad6 staining in cytoplasmic and nuclear subcompartments of both singly nucleated (A) and multinucleated (D and G) cells. B, E, and H demonstrate proliferative potential of singly and multinucleated HHR6B-overexpressing cells, and C, F, and I show colocalization of Rad6 and PCNA. Note the absence of colocalization of Rad6 and PCNA in C (short white arrow) indicating the presence of cell cycle-regulated alterations in Rad6-PCNA interactions. Also, note the presence of nuclei of various sizes (F and I) and elimination of distinct nuclear entity from a multinucleated cell (long white arrow). Panels J–L represent Rad6 and PCNA localization in parental MCF10A cells. Note colocalization of Rad6 with PCNA in a dividing MCF10A cell. Bars, 10 µm.
), which coincides with its colocalization with PCNA (Fig. 4c)