ISSN 0026-8933, Molecular Biology, 2007, Vol. 41, No. 1, pp. 77–85. © Pleiades Publishing, Inc., 2007. Original Russian Text © E.A. Anedchenko, N.P. Kisseljova, A.A. Dmitriev, F.L. Kisseljov, E.R. Zabarovsky, V.N. Senchenko, 2007, published in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 1, pp. 86–95.
CELL MOLECULAR BIOLOGY UDC 575:599.9
Tumor Suppressor Gene RBSP3 in Cervical Carcinoma: Copy Number and Transcription Level E. A. Anedchenkoa, d, N. P. Kisseljovab, A. A. Dmitrieva, F. L. Kisseljovb, E. R. Zabarovskyc, and V. N. Senchenkoa a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia; e-mail:
[email protected] b Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, 115478 Russia c MTC, Karolinska Institute, Stockholm, 17177 Sweden d Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, 119992 Russia Received May 30, 2006 Accepted for publication August 7, 2006
Abstract—Real-time PCR was used to quantitate structural and functional aberrations in the tumor suppressor gene RBSP3 (3p.21) in papillomavirus-positive cervical carcinoma (CC). RBSP3 deletions were found in 19 of 45 tumor specimens (42%). The frequency of deletions was reliably higher in tumors with metastases to regional lymph nodes (64%) than in tumors without metastases (32%, P < 0.05). In several cases, RBSP3 amplification was detected. Changes in RBSP3 copy number were observed in 51% (23/45) of the tumor specimens. The RBSP3 transcription level was decreased in 21 of 33 tumors (64%), this event being detected more frequently in tumors with (83%) than without metastases (52%, P < 0.05). In some tumors, RBSP3 mRNA levels were increased. Changes in RBSP3 mRNA level were observed in 79% (26/33) of the CCs. Comparative analysis of the data demonstrated that a decrease in RBSP3 mRNA level was a result of deletions only in 23% of the specimens, while it was not associated with any changes in the gene copy number in 36% of the cases. More unusual combinations of events were an unchanged or even increased mRNA level in the presence of deletions or an increased mRNA level in the absence of changes in gene copy number. It was assumed that deregulation of RBSP3 expression is associated with the progression of CC, that there is more than one mechanism of RBSP3 inactivation, and that RBSP3 has other functions in addition to pRb dephosphorylation. DOI: 10.1134/S0026893307010116 Key words: cervical carcinoma, human chromosome 3, tumor suppressor gene RBSP3, gene copy number, transcriptional level, tumor progression
INTRODUCTION
STS marker NLJ-003 located in the short arm of human chromosome 3 (region AP20, 3p.21.3), [3, 6, 7]. NLJ-003 is in the promoter region of the tumor suppressor gene RBSP3 (RB1 serine phosphatase from human chromosome 3, or CTDSPL, HYA22, SCP3, etc.; GenBank acc. nos. A575644 and AJ5755645). This 123-kb gene codes for a nuclear protein of 340 amino acids that belongs to the family of small CTD-serine phosphatases (Carboxy-Terminal Domain). The product of RBSP3 is capable of suppressing tumor growth in cell cultures and mice; exogenous RBSP3 expression in cells is accompanied by a decrease in phosphorylated pRb. It has been proposed that RBSP3 is involved in the negative regulation of the cell cycle by pRb dephosphorylation [7].
Cervical carcinoma (CC) is the second (to breast cancer) most common female malignancy. Etiology of CC is attributed to certain types of papillomaviruses (HPV type 16, 18, and related so-called high-risk viruses). It is known that the transformed phenotype of HPV-positive tumors depends on the expression of the HPV oncogenes Ö6 and Ö7, whose major function is inactivating tumor suppressors p53 and pRb, which are essentially involved in controlling cell proliferation and differentiation, genomic integrity, and apoptosis [1]. Genomic instability developing in the course of progression of HPV-positive CCs (as well as of other tumors) gives rise to various genetic defects. It has been shown previously that genetic changes in CC affect chromosomes 3p, 4q, 6p, 11q, and some others [2–5]. A high rate of deletions, amplifications, and point mutations has been detected in various epithelial tumors, including CC, by means of a universal NotI–
We have shown earlier that RBSP3 expression is considerably reduced in cell lines and in some primary epithelial tumors. Cases of enhanced expression and amplification of mutant gene variants have also been 77
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reported in tumors [7]. This character of expressional changes suggests bifunctionality of the gene. It cannot be ruled out that RBSP3 has both tumor suppressor and oncogenetic activities. The signal pathway p16ink4a–Cdk4/cyclinD1–pRb, where RBSP3 is most likely involved, is disrupted in most tumors because of inactivation of its various components. HPV-positive CCs differ from tumors of nonviral nature in how this pathway is inactivated: the product of E7 interacts with pRb, causing its inactivation and degradation [8–10]. The nearly complete absence of pRb in most cells of HPV-positive tumors, the supposed involvement of RBSP3 in pRb dephosphorylation, and possible bifunctionality of RBSP3 are the reasons to expect that changes in RBSP3 expression in CCs differ from those in other tumors. This led us to quantitatively analyze the RBSP3 copy number and transcription level in HPV-positive CCs. No such data have been published before. Our main method was real-time PCR, a technique that has lately become a common tool for quantitating gene expression, owing to its obvious advantages over traditional qualitative and semiquantitative methods, like Northern hybridization, RT–PCR, and others. EXPERIMENTAL Tissue specimens. Specimens of squamous CCs, adjacent morphologically normal tissues, and peripheral blood lymphocytes from patients with CC stages I–III (according to the classification of the International Federation of Gynecology and Obstetrics, FIGO) were obtained at the Blokhin Cancer Research Center. Tumor tissues were histologically typed and were separated into tumor and morphologically normal tissues in the Pathology Department of the Cancer Research Center. In all patients diagnosed with CC stage III, metastases in regional lymph nodes was confirmed histologically. Patients with CC stage I or II had no metastasis. As shown by PCR, RT–PCR, and, in several cases, by Southern blotting, a high-risk HPV genome (type 16 or 18) was present in all CC specimens. RNA and DNA from normal and tumor human tissues were isolated with guanidine isothiocyanate with subsequent ultracentrifugation through a cesium chloride cushion [11]. DNA from peripheral blood lymphocytes was isolated according to the following protocol. Leukocytes were suspended in buffer LST (29 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2). Then, an equal volume of 4 × TNLB (29 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 5% sucrose, 4% Np-40) was added, and the mixture was stirred gently and centrifuged at 3000 g for 10 min. The pellet was washed twice with LST–sucrose; combined with buffer TNE (0.01 M NaCl, 0.01 M TrisHCL, pH 8.0, 0.01 M EDTA), 1% SDS, and 100 µg/ml
proteinase K; and incubated overnight at 50°C. Following that, DNA was extracted by adding an equal volume of chloroform–isoamyl alcohol (24 : 1) and shaking the mixture for 5 min. After centrifugation, the upper phase was collected and the procedure repeated. Then DNA was precipitated with three volumes of iced ethanol and centrifuged at 8000 g at 4°C. The DNA pellet was washed with cold 75% ethanol on 1 × STE (0.1 M NaCl, 0.01 M Tris-HCl, pH 7.3, 1 mM EDTA), dried, and dissolved in bidistilled water. DNA and RNA samples obtained from CC specimens were adjusted to the same OD and controlled by electrophoresis in agarose gels. Synthesis of cDNA was done according to the recommendations of Applied Biosystems. The reaction mixture for reverse transcription contained 1 µg of RNA, 100 ng of hexanucleotide primers, 1 mM each dNTP, the reaction buffer (250 mM Tris-HCl, pH 8.3, 25°C, 250 mM KCl, 20 mM MgCl2, 50 mM DTT), and 200 units of M-MuLV reverse transcriptase (Fermentas, Lithuania). The reaction was performed in 20 µl for 10 min at 25°C, 60 min at 42°C, 10 min at 50°C and 10 min at 70°C. DNA and cDNA samples for real-time PCR. The study was performed with 76 DNA specimens (45 specimens from tumor tissues, stages I–III and 31 paired specimens from adjacent normal tissues, used as a reference norm) and 59 cDNA specimens (34 from tumors, stage I–III, and 25 matching reference norms). For DNA and cDNA samples with no matching reference norm available, calculations were performed using the threshold cycle Ct averaged over several reference norms. There were 31 pairs of DNA and cDNA samples obtained from the same tumor tissues. Primers and probes for real-time PCR. The RBSP3 copy number was evaluated using the forward and reverse primers 5'-CAGAGTGCGTGTGCCGACT-3' and 5'-ACAACTTCTCTGCGGGCG-3', and the probe 5'-CTGGCGGAGAGACTGGGAGCGA-3' (a 125-bp amplicon). For the control β-actin gene (ACTB), we used the forward primer 5'-GTGCTCAGGGCTTCTTGTCCTTT-3', the reverse primer 5'-TTTCTCCATGTCGTCCCAGTTGGT-3', and the probe 5'-AAGGATTCCTATGTGGGCGACGAGGCCCA-3' (a 160-bp amplicon). The RBSP3 transcription level was evaluated using the forward primer 5'-GCGAGAAAGCCTCCCAGTG-3', the reverse primer 5'-CCACCATTCTCCTCCACCAGT-3', and the probe 5'-CCACATTGTAATCACGGAAGCAGCAGA-3' (a 154-bp amplicon). For control GAPDH, we used the forward and reverse primers 5'-CGGAGTCAACGGATTTGGTC-3' and 5'-TGGGTGGAATCATATTGGAACAT-3' and the probe 5'-CCCTTCATTGACCTCAACTACATGGTTTACAT-3' (a 141-bp amplicon.). All probes carried the FAM dye on the 5' end and the Dabsyl quencher on the 3' end. MOLECULAR BIOLOGY
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The sensitivity of the method was tested with primers and a probe directed to the phosphofructo-2-kinase gene (PF2K): forward primer 5'-ATGCCCTGGCCAACTCA-3', reverse primer 5'-TGCGACTGGTCCACACCTT-3', and the probe 5'-FAM-TCAGTCCCAGGGCATCAGCTCCCTAMRA-3' [12]. The optimal primer and probe concentrations for the test and control genes were as follows: genomic RBSP3 primers, 150 nM; probe, 100 nM; ACTB primers, 200 nM; probe, 100 nM; expression RBSP3 primers, 350 nM; probe, 150 nM; GAPDH primers, 300 nM; probe, 150 nM. Real-time PCR was performed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The reaction efficiency was evaluated using a relative standard curve. Reactions were performed in 25 µl as described earlier [3]. Analysis of real-time PCR products. PCR products (4-µl aliquots) were analyzed in 1.8% agarose gels with 0.5 µg/ml ethidium bromide, using the M-50 marker (Izogen, Russia) to estimate the size of the amplified fragments. The nucleotide sequences of the amplicons were verified by sequencing in a 3730 DNA Analyzer automated sequencer (Applied Biosystems). Fragments were sequenced independently with forward and reverse primers, using a DYEnamic ET Terminator Cycler Sequencing kit (Amersham). All primers and probes were specific in real-time PCR; the lengths and nucleotide sequences of the amplicons were as expected. Data analysis. Reactions were performed in the relative quantification mode, using the software of Applied Biosystems, and the data were imported in the form of a text file in Microsoft Excel for mathematical analysis. The relative gene copy number RDNA and the relative transcription level RcDNA were computed as a ratio of the target DNA or cDNA copy number to the control copy number in the tumor (T) and norm (N); their relation to the basic parameters of real-time PCR, the threshold cycle Ct and the reaction efficiency A, is given by equation Ct
test gene
( 1 + E test gene ) ------------------------------------------------------comtrol gene Ct ( 1 + E control gene ) norm R = ----------------------------------------------------------------------. test gene Ct ( 1 + E test gene ) ------------------------------------------------------comtrol gene Ct ( 1 + E control gene ) tumor
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interval from 2–∆∆Ct + e to 2–∆∆Ct – e, where e is the standard deviation from ∆∆Ct: n
∑ e =
n
( xi – x )
∑x
2
i=1
---------------------------- , where (n – 1)
i
i=1
x = ------------ , n
i = 1, 2, …, n, and n is the number of times the reaction was repeated (in our case, n = 3). The deviation for the sum of values was calculated as k
e =
∑e
2 j ,
j = 1, 2, …, k,
j=1
where k is the number of values being summed. The efficiency of real-time PCR for RBSP3 and the control genes in tumors and normal tissues was calculated based on the slope of relative standard curves, following the Applied Biosystems protocol. Sensitivity of real-time PCR was controlled by determining the sexual origin of DNA samples. Realtime PCR of PF2K was performed on 30 DNA probes, 15 male and 15 female, with the ACTB control. Reproducibility of real-time PCR. The reaction was performed three times in every run and was repeated with some specimens in separate experiments. Only those values whose standard deviations did not exceed 10% of the mean were used for the data analysis. When real-time PCR was repeated in different experiments, the values were close enough to fall within overlapping intervals calculated for the deviations. Statistical analysis of the data was performed using the Student–Fisher method. Results were considered valid and differences significant at P < 0.05. RESULTS Sensitivity and Validity of Real-Time PCR
When the reaction efficiencies for the test and control genes have close values, a so-called comparative, or ∆∆C t, method is used. When E values are close to 100%, R = 2–∆∆Ct, where ∆∆Ct = ∆Ct(T) – ∆Ct(N). In our case, ∆Ct = CtRBSP3 – CtAëTB for DNA and ∆Ct = CtRBSP3 – CtGAPDH for cDNA. R = 2–∆∆Ct falls within the MOLECULAR BIOLOGY
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It is known that the phosphofructo-2-kinase gene (PF2K) is on the X chromosome. Figure 1 represents the results of real-time PCR of PF2K and ACTB. The values of ∆Ct = Ct(PF2K) – Ct(ACTB) for the 30 specimens clearly fell into two groups. For 15 male probes, ∆Ct was 6.01 ± 0.40; and for 15 female samples, 4.91 ± 0.44. Normalized to the ACTB copy number, the ratio of the PF2K copy number in the female and male probes was NF/NM = 2–∆∆Ct = 2.14 (2.08– 2.20); i.e., the difference was twofold. Thus, the sensitivity of real-time PCR was sufficient to reliably dis-
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∆Ctå = 6.0 ± 0.4 ∆CtF = 5.0 ± 0.4
7 6 ∆Ct
5 4 3 2 1 0
1
2
3
4
5
6
7 8 9 Specimen
10
11
12
13
14
15
Fig. 1. Copy number of the X-chromosomal phosphofructo-2-kinase gene (PF2K) relative to the control β-actin gene (ACTB) in male and female DNA specimens.
tinguish one from two genomic copies of the nucleotide sequence in question. Choosing Control Genes for Normalization the Real-Time PCR Data In the present study, we chose as a reference the genes for β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), commonly used as controls. In tumor and normal tissues, the variation of the R value for ACTB and GAPDH, reflecting the initial amount of DNA or cDNA in the reaction, was no more than 1.5-fold (P < 0.05), indicating that the variation of the copy number and transcription level of these genes in cervical tissues was acceptable for normalization of real-time PCR data. According to the GeneCards and Pseudogene.Org databases, the human genome contains 52 GAPDH pseudogenes. Our primers to GAPDH, as well as primers offered by Applied Biosystems, can hybridize with several pseudogenes. However, contaminating DNA accounted for 0.003 to 0.6% in paired RNA specimens obtained from normal and tumor tissues and had hardly any effect on the results of real-time PCR. Therefore, the existence of pseudogenes did not actually affect the results. Reaction Efficiency for RBSP3 and the Control Genes The efficiency of all reactions for RBSP3 and the control genes in CCs and normal tissues was about 100% (Table 1). Hence, the computations were performed with the equation R = 2–∆∆Ct.
Reference DNA and cDNA Samples from Normal Tissue Since no paired reference norms were available for some tumor specimens, the relative gene copy number in tumor DNA was computed in relation to different reference specimens: to the matching reference norms whenever available or, otherwise, to the reference norm averaged over 14 patients. The average of ∆Ct = Ct(RBSP3) – Ct(ACTB) for three specimens of lymphocyte DNA served as an additional reference. In most cases, RDNA values were similar for different reference specimens. In several matching reference norms, however, we observed a two- to fourfold reduction in RBSP3 copy number in comparison to lymphocyte DNA, which may implicate RBSP3 in some early events occurring in tissues adjacent to the tumor; hence, these specimens were considered inappropriate as references. Reverse transcription was always performed with the same RNA amount; for this reason, the values ∆Ct = Ct(RBSP3) – Ct(GAPDH) were close for samples of conventionally normal cDNA. This justifies the use of average normal values as reference for those tumor cDNAs where no matching norms were available. Changes in RBSP3 Copy Number in CC Changes in RBSP3 copy number were quantitated using genomic primers to the promoter region and the short 200-bp intron between exons 1 and 2. It is generally accepted that RDNA values of 0.7–1.5 correspond to an unchanged DNA copy number. Taking into account a slight variation in the copy numbers of the control genes, we considered at least 1.5-fold RDNA changes (increases or decreases). The results are presented in Table 2. MOLECULAR BIOLOGY
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RBSP3 IN CERVICAL CARCINOMA Table 1. Reaction efficiencies calculated from relative standard curves as E(%) = (10–1/tanα – 1) × 100 Source
Gene
DNA, norm
RBSP3 ACTB DNA, tumor RBSP3 ACTB DNA, lymphocytes RBSP3 ACTB cDNA, norm RBSP3 GAPDH cDNA, tumor RBSP3 GAPDH
tanα
Efficiency, %
–3.23 –3.32 –3.23 –3.10 –3.23 –3.34 –3.32 –3.32 –3.33 –3.32
103 99.5 103 99 103 99 99.5 99.5 100 99.5
In CC specimens, we observed both reductions (deletions) and increases (amplifications) in RBSP3 copy numbers. Most commonly (in 42% of the cases), we detected a decrease in gene copy number, typically two- to threefold. In three specimens, the decrease was three- to ninefold, indicating homozygous deletions. Comparing tumors with metastases in regional lymph nodes (stage III) to tumors without metastasis (stages I and II), we found that the frequency of deletions was reliably higher in advanced tumors (9/14 vs. 10/31, P < 0.05, Table 2). Increases in gene copy number (1.5- to 3-fold) were detected in 4 out of 45 specimens (9%), all being of stages I and II. Altogether, changes in relative gene copy number were found in 51% (23 of 45) tumor DNA specimens. In sammary, we detected deletions and rare amplifications of RBSP3 in CC and found an association between an increased frequency of RBSP3 deletions and metastatic progression of the tumor. There was no correlation between the tumor size and the frequency of deletions. Changes in RBSP3 Transcription Level in CC To quantitate the changes in RBSP3 transcription level, we used primers to the coding region of the gene: the forward primer to the boundary of exons 2
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and 3, and the reverse primer and the probe to exon 3. In comparison to normal tissues, both decreased and increased mRNA levels were observed in CCs (Table 3). In CC stage I, the frequency of a decrease in mRNA level was relatively high (33%); at stages II and III, it reached 66 and 83%, respectively. The extent of a decrease also associated with tumor progression: it was up to fivefold at stages I and II and up to 20-fold at stage III. The frequency of a decrease in transcription level was 52% (11/21) in tumors without metastasis (stage I–II) and 83% in CC stage III, with metastasis to regional lymph nodes; i.e., a decrease in RBSP3 transcription level significantly correlated with metastatic progression of the tumor (P < 0.05). There was no correlation between the tumor size and the frequency of a decrease in RBSP3 transcription level. In 5 of 33 CCs (15%, all stages I–II), transcription was increased two- to fourfold. Altogether, changes in RBSP3 transcription level were found in 79% (26/33) of CCs, including 64% (21/33) of specimens with a decreased transcription level. Thus, the frequency of a decrease in RBSP3 transcription level in CC was high, although an increased transcription level was observed in some cases, and the frequency of a decrease in RBSP3 transcription level correlated with metastatic progression of the tumor. Comparison of Changes in RBSP3 Copy Number and Transcription Level We used 31 pairs of DNA and cDNA samples (9/I, 12/II, 10/III) obtained from the same tumor tissue to compare the concomitant changes in RBSP3 gene copy number and transcription level. Figure 2 and Table 4 represent different combinations of parallel changes/lack of changes in gene copy number and mRNA level. A decrease in mRNA level was the most common event observed (58% of CCs). In 23% of these cases, such a decrease was associated with RBSP3 deletions, while the RBSP3 copy number was unchanged in the other 36% (P < 0.05, groups 1 and 2, Table 4). While the RBSP3 copy number decreased only two- to threefold in most CCs, a decrease in mRNA level was more profound, reaching a 20-fold
Table 2. Frequency of changes in RBSP3 copy number in cervical carcinomas Clinical stage I II I + II (without metastases) III (with metastases) I + II + III MOLECULAR BIOLOGY
Frequency of changes in copy number, % deletion, RDNA ≤ 0.7
amplification, RDNA ≥ 1.5
total changes
27 (4/15) 38 (6/16) 32.3 (10/31) 64 (9/14) (P < 0.05) 42 (19/45) (P < 0.05)
20 (3/15) 6 (1/16) 12.9 (4/31) 0 (0/14) 9 (4/45)
47 (7/15) 44 (7/16) 45.2 (14/31) 64 (9/14) 51 (23/45) (P < 0.05)
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Table 3. Frequency of changes in RBSP3 mRNA level in cervical carcinomas Clinical stage I II I + II (without metastases) III (with metastases) I + II + III
Frequency of changes in mRNA level, % deletion, RDNA ≤ 0.7
amplification, RDNA ≥ 1.5
33.3 (3/9) 66 (8/12) 52.4 (11/21) (P < 0.05) 83 (10/12) (P < 0.05) 64 (21/33) (P < 0.05)
rate in CC stage III. Only in group 2 were the simultaneous events (a decreased transcription level and deletions, Table 4) nearly three times more frequent in advanced CCs than in tumors without metastases (40% vs. 14%). In 16% of CCs, neither RBSP3 mRNA level nor gene copy number was changed, indicating that malignancy can develop independently from any changes in RBSP3 copy number or gene regulation. Other combinations of changes in RBSP3 copy number and expression, e.g., an unchanged or increased mRNA level in the presence of deletions, or an increased mRNA level in the absence of changes in gene copy number (groups 4–6, Table 4), represent
33.3 (3/9) 17 (2/12) 23.8 (5/21) 0 (0/12) 15 (5/33)
total changes 67 (6/9) 83 (10/12) 76.2 (16/21) 83 (10/12) 79 (26/33) (P < 0.05)
rare variants of RBSP3 deregulation in CC (3–13% of cases). DISCUSSION Specific Features of Real-Time PCR Real-time PCR has lately become the main technique of quantitating gene expression [13, 14]. Despite the rapid development and wide practical use of the method, the validity and reproducibility of the results are still widely discussed [15–17]. That is why a number of preliminary tests must be performed. We determined the accuracy of measurements, checked the suitability of the chosen control genes, established
10
R
1
0.1
Stage I 0.01
Stage II
Stage III
235 289 414 426 451 237 257 407 415 427 455 254 434 439 448 458 284 413 424 436 201 239 261 411 418 454 234 409 438 445 450 Copy number Transcription level Fig. 2. Changes in RBSP3 copy number and transcription level in CCs compared to normal tissue. MOLECULAR BIOLOGY
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Table 4. Frequency of concomitant changes in RBSP3 copy number and transcription level in cervical carcinoma Simultaneous events Group
Frequency, % stages I + II, without metastases
stage III, with metastases
Decreased
38 (8/21)
30 (3/10)
Deleted
Decreased
14.3 (3/21)
40 (4/10)
Unchanged Unchanged Deleted Deleted
Unchanged Increased Unchanged Increased
14.3 (3/21) 19.1 (4/21) 9.5 (2/21) 4.8 (1/21)
20 (2/10) 0 10 (1/10) 0
gene copy number
mRNA level
1
Unchanged
2 3 4 5 6
close efficiencies of all reactions compared, and proved the reproducibility of the results obtained using different reference specimens (norms), as well as the statistical significance of changes in the parameters under study. Many studies have been devoted to the problem of choosing the control for normalization of quantitative data [16, 17]. Most often, housekeeping genes are used, although any gene with a relatively stable transcription level or DNA/cDNA copy number would be acceptable. Actually, the decision concerning the acceptable variation of the control gene depends on the accuracy required [16]. In our specimens, ACTB copy number and GAPDH transcription level did not vary more than 1.5-fold, which was quite satisfactory. Our choice was the comparative ∆∆Ct method, since we were mainly interested not in the absolute values but in the changes in gene copy number and transcriptional level in a tumor sample compared to norm. We showed that the method clearly discriminated between one and two copies of a nucleotide sequence. Should there be a need in detecting and interpreting smaller differences that was not the purpose of the present study, the validity of the data is to be proved additionally. Possible Pathways of Inactivation/Deregulation of RBSP3 Expression in CC To assess the association between the changes in the copy number and transcription of RBSP3 at different CC stages, we quantified and compared these changes and related them to tumor progression. In HPV-positive CCs, as in other tumors, we observed both repression (in most specimens) and activation (in a few cases) of RBSP3 expression. Decreased transcription level. The high frequency of a decrease in RBSP3 transcription (64% of cases) and an increase of the frequency (up to 83%) in advanced tumors suggest that this change is an imporMOLECULAR BIOLOGY
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I + II + III 35.5 (11/31) (P < 0.05) 22.6 (7/31) (P < 0.05) 16.1 (5/31) 12.9 (4/31) 9.7 (3/31) 3.2 (1/31)
Specimen
289, 424, 201, 237, 407, 411, 454, 455, 434, 450, 458 451, 261, 418, 409, 438, 439, 448 414, 436, 257, 234, 254 235, 413, 239, 427 426, 415, 445 284
tant event in CC progression. In 36% of the cases, the transcription level was decreased without any changes in gene copy number; in 23% of the cases, the decrease was due to deletions. One gene copy was lost in most tumors irrespective of the clinical stage. The greatest (nearly 20-fold) loss in RBSP3 mRNA level was observed in stage III CCs. This character of changes implies some further transcriptional repression pathways other than deletions. It is known that methylation of CpG islands in promoters and first exons is a common mechanism inactivating suppressor genes in tumors [18]. Since the RBSP3 promoter contains a CpG-island, we can suppose that, in the absence of changes in gene copy number, regulation of RBSP3 expression involves methylation; this could also explain a profound decrease in mRNA level when changes in the gene copy number are small. However, further studies are required to verify this hypothesis. Enhanced RBSP3 transcription. Some more unusual combinations of simultaneous events were observed (Table 4). In 5 of 31 tumor specimens (15%), RBSP3 transcription was increased, while the gene copy number was unchanged in four of them and one gene copy was lost in the fifth specimen. We have previously demonstrated for epithelial tumors of other locations that increased levels of the RBSP3 mRNA are always caused by transcription of a mutant gene. A high rate of missense and nonsense mutations in RBSP3 has been observed both in tumor biopsy specimens and in tumor cell lines [7]. De novo mutations arising during the cell cycle reduce the RBSP3 suppressor activity in vitro and in vivo (Kashuba et al., submitted article). An increase in RBSP3 transcription level may promote protein inactivation/loss of function. It is likely that increased levels of RBSP3 transcription in CC are due to mutations. There are no quantitative data on RBSP3 expression. Using real-time PCR, we have previously evaluated the RBSP3 transcription level in 12 epithelial tumor cell lines and in primary tumor specimens of
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renal (4), breast (2), and ovarian (2) carcinomas [7]. The RBSP3 transcription level was decreased more than 20-fold in 11 cell lines, including two lines derived from CC, and up to tenfold in three of the eight tumor specimens (in two renal and one breast carcinoma). In one lung cancer cell line, RBSP3 expression was enhanced nearly 40-fold. Our present work continues this research in more detail. For the first time, RBSP3 transcription level and gene copy number were evaluated in the same CC specimens. Probable Functions of RBSP3 There is little information on the functions of RBSP3. As mentioned above, exogenous RBSP3 expression in MSF-7 breast cancer cells is accompanied by a loss of inactive C-phosphorylated pRb, suggesting that RBSP3 phosphatase participates in pRb activation and in the negative regulation of the cell cycle [7]. Under this consideration, repression of RBSP3 in tumor tissues should promote uncontrolled cell proliferation. However, it is well known that pRb is undetectable immunohistochemically in most HPVpositive CC cells, because viral oncoprotein E7 is capable of binding pRb and Rb-like proteins and enhancing their degradation by ubiquitination [8–10]. The decrease in RBSP3 mRNA level we observed in more than 60% of HPV-positive CCs implies that RBSP3 not only participates in pRb dephosphorylation but also possesses some other tumor suppressor activity, which, when repressed, promotes CC progression. We have recently demonstrated that RBSP3 binds with RNA polymerase II (CTD RNAPII) in vitro and in vivo (Kashuba et al., unpublished results). It is known that dephosphorylation affects the activity of this enzyme (e.g., see [19]). However, the potential role of the RBSP3 interaction with RNA-polymerase II in carcinogenesis has not been established yet. Using the modern highly sensitive method of realtime PCR, we were the first to evaluate the transcription level and copy number of the tumor suppressor gene RBSP3 in CC. We observed a high frequency of a decrease in mRNA level (64%), a high frequency of genomic deletions (42%), and a correlation of the frequency of deletions and decreased mRNA levels with tumor progression. The comparative analysis of the simultaneous events on the genomic and mRNA levels in the same tumor specimens demonstrated that a decrease in RBSP3 transcription level was mainly associated with deletions or no change in gene copy number. We observed different combinations of simultaneous events, which suggests more than one mechanism of RBSP3 inactivation. Moreover, our results suggest that RBSP3 has some yet unknown functions other than pRb dephosphorylation. Further research should evaluate the decrease in RBSP3 transcription level as a diagnostic and prognostic molecular marker for CC.
ACKNOWLEDGMENTS We are grateful to L.L. Kisselev for useful recommendations and discussion of the manuscript, to O.V. Sakharova and T.T. Kondratjeva for valuable remarks, and to N.Yu. Oparina and G.S. Krasnov for their help in primer design. This work was supported by the Russian Foundation for Basic Research (project nos. 05-04-49408, 04-0449074, 04-04-08154) and INTAS (grant no. 03-51-4983). REFERENCES 1. Zur Hausen H. 2002. Papillomaviruses and cancer: From basic studies to clinical application. Nature Rev. Cancer. 2, 342–350. 2. Zabarovsky E.R., Lerman M.I., Minna J.D. 2002. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene. 21, 6915–6935. 3. Senchenko V., Liu J., Braga E., Mazurenko N., Loginov W., Seryogin Y., Bazov I., Protopopov A., Kisseljov F.L., Kashuba V., Lerman M.I., Klein G., Zabarovsky E.R. 2003. Deletion mapping of cervical carcinomas using quantitative real-time PCR identifies two frequently affected regions in 3p21.3. Oncogene. 22, 2984–2992. 4. Chatterjee A., Pulido H., Koul S., Beleno N., Perilla A., Posso H., Manusukhani M., Murty V.S. 2001. Mapping the sites of putative suppressor genes at 6p25 and 6p21.3 in cervical carcinoma: Occurrence of allelic deletions in precancerous lesions. Cancer Res. 61, 2119–2123. 5. Mazurenko N.N., Beliakov I.S., Bliyev A.Yu., Guo Z., Hu X., Vinokourova S.V., Bidzieva B.A., Pavlova L.S., Ponten J., Kisseljov F.L. 2003. Genetic alterations at chromosome 6 associated with cervical cancer progression. Mol. Biol. 37, 472–481. 6. Senchenko V.N., Liu J., Loginov W., Bazov I., Angeloni D., Seryogin Y., Ermilova V., Kazubskaya T., Garkavtseva R., Zabarovska V.I., Kashuba V.I., Kisselev L.L., Minna J.D., Lerman M.I., Klein G., Braga E.A., Zabarovsky E.R. 2004. Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung, and breast carcinomas. Oncogene. 23, 5719–5728. 7. Kashuba V.I., Li J., Wang F., Senchenko V.N., Protopopov A., Malyukova A., Kutsenko A.S., Kadyrova E., Zabarovska V.I., Muravenko O.V., Zelenin A.V., Kisselev L.L., Kuzmin I., Minna J.D., Winberg G., Ernberg I., Braga E., Lerman M.I., Klein G., Zabarovsky E.R. 2004. RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies. Proc. Natl. Acad. Sci. USA. 101, 4906–4911. 8. Fieder M., Muller-Holzner E., Viertler H.P., Widschwend ter A., Laich A., Pfister G., Spoden G.A., JansenDurr P., Zwerschke W. 2004. High level HPV-16 E7 oncoprotein expression correlates with reduced pRb-levels in cervical biopsies. FASEB J. 10, 1120–1122. 9. Tringler B., Gup C.J., Singh M., Groshong S., Shroyer A.L., Heinz D.E., Shroyer K.R. 2004. Evaluation of p16 and Rb expression in cervical squamous and grandular neoplasia. Human Pathol. 35, 689–696. MOLECULAR BIOLOGY
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