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Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To date, at least six groups of initiation proteins, including ORC (Orc1p through -6p; Refs. 3, 4, 5, 6, 7 ), Noc3p (8) , Cdc6p (1 , 9, 10, 11, 12) , Cdt1p (13, 14, 15, 16) , MCM (Mcm2p- through -7p; Refs. 17, 18, 19, 20 ), and Cdc45p (21, 22, 23) , are known to be required for eukaryotic DNA replication. These proteins are conserved in eukaryotes, and homologues of most of them have been identified in model organisms from yeast to humans. To test the idea that inhibiting the expression of replication-initiation proteins can stop cancer cell growth, we have designed, screened, and tested antisense ODNs4 and siRNA molecules targeted to three human DNA replication-initiation genes, hCdc6, hMcm2, and hCdc45. We found that selective antisense ODNs and siRNAs not only significantly reduced the mRNA and protein levels of the target gene products, thus stopping DNA replication and cell proliferation, but also resulted in apoptosis of both p53-positive and -negative cancer cells. Furthermore, silencing of these genes does not cause death of cells derived from normal tissues.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All ODNs were custom-synthesized and purified to 
99% (MGW Biotech, Ebersberg, Germany), and the 16 phosphorothioate-end-modified antisense ODNs and control ODNs were further ethanol-precipitated twice to remove small-molecule impurities. Synthetic siRNAs (Dharmacon, Lafayette, CO) were further ethanol-precipitated twice. Following are the regions of the genes targeted by the 16 end-modified antisense ODNs and 8 siRNAs, as specified by the nt numbers of cDNA sequence entries (hCdc6, U77949; hMcm2, NM_004526; and hCdc45, AF053074) in databases: The antisense ODNs (C6-, targeted to hCdc6; M2-, targeted to hMcm2; C45-, targeted to hCdc45) were as follows: C6-30 (nt 1006–1025), C6-33 (nt 1163–1181), C6-35 (nt 1231–1246), C6-39 (nt 1550–1567), M2-33 (nt 1686–1705), M2-34 (nt 1716–1731), M2-47 (nt 2330–2346), M2-61 (nt 3268–3283), C45-8 (nt 455–469), C45-18 (nt 874–893), C45-22 (nt 1040–1059), and C45-30 (nt 1200–1215). The siRNAs (siC6., targeted to hCdc6; siM2., targeted to hMcm2; siC45., targeted to hCdc45): siC6.1 (nt 1234–1254), siC6.2 (nt 1085–1105), siC6.3 (nt 2509–2529), siM2.1 (nt 643–663), siM2.2 (nt 2727–2747), siM2.3 (nt 2804–2824), siC45.1 (nt 1189–1209), and siC45.2 (nt 460–480). The sequences of the mismatched control ODNs and RNAs that appear in the figures are as follows: C6-35-mm, 5'-AAGATGGGTAGGTCAA-3'; M2-47-mm, 5'-TCCCTCAGGTGGAAGCG-3'; C45-30-mm, 5'-AAGGAGTTGTCTCTCC-3'; siC6.1 mm (the sense strand; same for other control RNAs), 5'-AATTTTCCGCCTTATACCAGA-3'; siC6.2 mm, 5'-AAGACCGGTAGGTTTAGCACA-3'; siM2.1 mm, 5'-AAGGAGCGCATCTCCGACATG-3'; and siC45.1 mm, 5'-AAGGATGGCTCGGGAACGGAC-3'.
Cell Culture, Transfection, and Cell Viability Assay.
All culture media and transfection agents were from Invitrogen (Carlsbad, CA). Cells were grown in various media supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. HeLa (cervical adenocarcinoma), Hone1 (nasopharyngeal cancer), T-Tn (esophageal cancer), and HepG2 (hepatocellular carcinoma) cells were grown in DMEM. NCI-H446 (small cell lung carcinoma), NCI-H460 (large cell lung carcinoma), Chang (from liver; a gift from M. Lung, Hong Kong University of Science and Technology, Hong Kong, China, who obtained the cells from R. S. Chang, University of California at Davis, Davis, CA, who generated the line; the cells we used are tumorigenic, as determined by non-contact-inhibited growth and xenograft growth in nude mice; data not shown), BEL-7402 (hepatocellular carcinoma), and L-02 (normal human liver cells; Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Shanghai, China) were cultured in RPMI 1640. Hep3B (hepatocellular carcinoma; p53-/-) were maintained in MEM. One day before transfection, cells were trypsinized, diluted in growth medium, and seeded in 96-well plates. At 70–80% confluence, cells were transfected with a complex of ODNs (final concentrations during transfection, 0.7 µM for HeLa, Hone1, T-Tn, and Chang cells and 1.0 µM for other cells) and 0.7 µl (for HeLa, T-Tn, and Chang cells) or 1.0 µl (for other cells) of Lipofectamine 2000 (Invitrogen) in 100 µl of OptiMEM medium for 4 h. Cells were then incubated in FBS-containing growth medium for 44 h, before WST-1 assays were performed as described previously (26)
. Counted numbers of cells were used to construct the standard curves. siRNAs were transfected into cells at 40–50% confluence with a complex of siRNA (final concentration during transfection, 100 nM) and 1.0 µl of Oligofectamine (Invitrogen) in 150 µl of OptiMEM medium for 18 h. The cells were then incubated in FBS-containing growth medium for 54 h before the WST-1 assay.
Rescue by the Silently Mutated hCdc6 Gene.
The sense sequence targeted by C6-35-as was changed from 5'-TTGAACTTCCCACCTT-3' to 5'-CTCAATTTTCCGCCGT-3'. The silently mutated and wild-type hCdc6 genes were cloned into pcDNA3.1/Zeo(-) (Invitrogen) to obtain pC6SM and pC6WT, respectively. Cells at 80–90% confluence were transfected with 0.9 µg of pcDNA3.1/Zeo(-), pC6WT, or pC6SM together with 0.2 µg of pEGFP-N1 (Clontech, Palo Alto, CA) in 6-well plates with use of Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. Transfected cells were trypsinized, split, and reseeded in growth medium, incubated for 12 h, and then further treated with Lipofectamine 2000, C6-35-mm, or C6-35-as or left untreated for normalization. Cells were collected 48 h post-ODN transfection, and GFP-positive cells were counted under a fluorescence microscope.
RT-PCR and Western Blotting.
Total RNA was isolated with use of TRIzol reagent (Invitrogen) at 0 h (for ODNs) or 6 h (for siRNAs) post-transfection. Total RNA (100 ng) was used for cDNA synthesis using First Strand cDNA Synthesis Kit (MBI, Ramat Hasharon, Israel). PCR coamplification of ß-actin with hCdc6, hMcm2, or hCdc45 was performed with 1 µl of cDNA and Taq polymerase in 25-µl reactions. The ratio of ß-actin primers to hCdc6, hMcm2, or hCdc45 primers was 1:10. The sequences of the RT-PCR primers were as follows: ß-actin, 5'-GATATCGCCGCGCTCGTCG-3' and 5'-GGGAGGAGCTGGAAGCAG-3'; hCdc6, 5'-GGCCAGGATGTATTGTACAC-3' and GGCCCGAATGTGTAAAGC-3'; hMcm2, 5'-TATTATCACTAGCCTCTCC-3' and 5'-GGTAGCGGGCAAAAGCTTG-3'; and hCdc45, 5'-CCAGGAGTTCCTTGCAGAC-3' and 5'-CAATCTAAATGAAAGCCAGTT-3'. The PCR program was as follows: 1 cycle of 95°C for 3 min followed by 35 cycles of 94°C for 30 s, 58°C for 3 s, and 72°C for 45 s and 1 cycle of 72°C for 5 min. For Western blotting, total proteins (15–20 µg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose. For hCdc6, cells were collected at 1 h (for ODNs) or 10 h (for siRNAs) post-transfection. For hMcm2 and hCdc45, cells were collected at 2 h post-transfection (for ODNs only), and proteins were treated with Lambda phosphatase (New England Biolabs, Beverly, MA) before SDS-PAGE. Antitubulin immunoblotting (data not shown) and Ponceau S staining were used as loading controls. All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), except for anti-hMcm2, which was from Becton Dickinson (Palo Alto, CA).
BrdUrd Incorporation.
Transfected cells grown on cover slips coated with poly-D-lysine (Sigma, St. Louis, MO) were incubated with 50 µM BrdUrd (Sigma) for 1 h at 37°C at 1 h (for ODNs) or 12 h (for siRNA) post-transfection. Cells were then fixed in 20 mM glycine (pH 2.0)–70% ethanol at room temperature for 45 min and incubated sequentially with anti-BrdUrd and antimouse IgG-FITC conjugates (Sigma), each for 1 h at 37°C, with three washes in PBS after each antibody incubation. BrdUrd incorporation was observed for at least 200 cells/sample under a fluorescence microscope.
Apoptosis Assays.
Apoptosis was assayed by use of the In Situ Cell Death Detection Kit with Fluorescein (TUNEL assay; Roche, Palo Alto, CA), the Annexin V-Cy3 Apoptosis Detection Kit (plus 6-CFDA; Sigma), and the caspase inhibitor Z-VAD-fmk (Enzyme Systems, Livermore, CA). For TUNEL assays, transfected cells grown on cover slips for 2 h (for ODNs) or 16 h (for siRNAs) post-transfection were fixed for 1 h in fresh 4% paraformaldehyde, rinsed in PBS, permeabilized for 2 min on ice in 0.1% Triton X-100–0.1% sodium citrate, and then labeled with FITC-dUTP and terminal transferase for 30 min. TUNEL-positive cells were observed under a fluorescence microscope. Annexin V assays were done according to the manufacturer’s instructions with some modifications. Cells on cover slips were stained with a solution of 6-CFDA and Annexin V–Cy3 at the same time points as in the TUNEL assays. Z-VAD-fmk (final concentration, 1 µM) was added to growth medium immediately after transfection, and the cells were incubated for another 48 h before the WST-1 assay.
Flow Cytometry.
Both floating and attached cells were collected and washed once with PBS. Cells were fixed in 70% ethanol for 1 h to overnight at -20°C, washed thoroughly with PBS, and then stained in 50 µg/ml RNase A, 0.1% Triton X-100, 0.1 mM EDTA (pH 7.4), and 50 µg/ml propidium iodide for 30 min at 4°C. Samples were analyzed with an FACSort instrument (Becton Dickinson).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because silencing of replication-initiation genes was expected to result in inhibition of DNA replication and thus of cell proliferation, the 170 antisense ODNs were first individually subjected to initial screening for their abilities to inhibit cell proliferation in human cell lines derived from liver, esophageal, and cervical cancers. Sixteen of the antisense ODNs were initially considered active and chosen for further analysis (see below) because each inhibited cell growth by 50–70% (i.e., viable cell numbers were 30–50% compared with untreated cells) in all of the cell lines tested, whereas the transfection agent alone inhibited cell growth by 20% (data not shown), as measured by the WST-1 cell viability assay (26)
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Selected Antisense ODNs and siRNAs Can Inhibit Target Gene Expression.
To investigate the effects on inhibition of target gene expression, DNA replication and cell proliferation, the 16 ODNs that showed antiproliferation activities in the initial screening were modified with phosphorothioate linkages at both the 5' and 3' ends to increase resistance to exonucleases. Similarly modified sense and mismatched ODNs were used as the controls. The mismatched ODNs had the same base compositions of the corresponding antisense ODNs, but one in every five nucleotides on average in each ODN was changed. Possible inhibition of target gene expression was analyzed in several human cancer cell lines (Chang, HepG2, Hep3B, HeLa, and Hone1) and normal human liver L-02 cells (27
, 28)
; we also confirmed that growth of L-02 cells is contact-inhibited (data not shown). Similar levels of transfection (90% as measured by using an FITC-labeled ODN, C6-35-as; data not shown) for different cell lines were obtained by optimizing the amounts of the transfection agent and ODNs. Total RNA and proteins from the untreated cells and cells treated with the transfection agent alone or transfected with the antisense or control ODNs were analyzed by RT-PCR (Fig. 1, A–C)
and Western blotting (Fig. 1, D–F)
, respectively. For RT-PCR, each RNA sample was analyzed for the mRNA of two other replication-initiation proteins in addition to the target gene and the internal control ß-actin gene was coamplified with each of the three replication-initiation genes.
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and L-02 cells (Fig. 1B)
, for example, C6-35-as targeted to hCdc6 lowered the level of hCdc6 mRNA (Fig. 1, A and B
, Lane 5, top panels), but not the mRNA levels of two other replication-initiation proteins, hMcm2p and hCdc45p, in the same cells transfected with C6-35-as (Fig. 1, A and B
, Lane 5, middle and bottom panels). Similarly, M2-47-as targeted to hMcm2 diminished the level of hMcm2 mRNA (Fig. 1, A and B
, Lane 8, middle panels) without affecting the levels of the mRNAs encoding hCdc6p and hCdc45p (Fig. 1, A and B
, Lane 8, top and bottom panels), and C45-30-as targeted to hCdc45 reduced the level of hCdc45 mRNA (Fig. 1, A and B
, Lane 11, bottom panels) but did not affect the levels of hCdc6 and hMcm2 mRNA (Fig. 1, A and B
, Lane 11, top and middle panels). The transfection agent (Lane LF) alone and the sense (Lane ss) and mismatched (Lane mm) control ODNs did not noticeably alter the expression levels of the genes analyzed (Fig. 1, A and B)
. Similar results were obtained for all other cell lines tested (data not shown). In addition, six other antisense ODNs, C6-60-as, M2-61-as, M2-33-as, C6-33-as, M2-34-as, and C45-18-as, also significantly reduced (by 
60%) the mRNA levels of the corresponding target genes (Fig. 1C)
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As the mRNA levels were specifically reduced by the antisense ODNs, the protein levels of the target genes were also correspondingly lowered (Fig. 1, D–F)
. For example, as shown in Fig. 1D
, the level of hCdc6p (top panels), but not of hMcm2p (middle panels) were reduced by C6-35-as (Lanes 3, 7, and 11) in three cell lines, whereas the transfection agent (Lanes 2, 6, and 10) and C6-35-mm (Lanes 4, 8, and 12) had no effect. Similarly, M2-47-as, but not the transfection agent or M2-47-mm, reduced the hMcm2p level (Fig. 1E)
, and C45-30-as, but not the transfection agent or C45-30-mm, lowered the hCdc45p level (Fig. 1F)
. Together, these data suggest that the antisense ODNs effectively and specifically inhibited the expression of the corresponding target genes.
We also used a different gene-silencing strategy, RNA interference. Three siRNAs (siC6.1, siC6.2, and siC6.3) targeted to hCdc6, three (siM2.1, siM2.2, and siM2.3) to hMcm2, and two (siC45.1 and siC45.2) to hCdc45 were designed based on general strategies (29)
, except that siC6.1 and siC45.1 were targeted to areas overlapping with the target sites of two active antisense ODNs, C6-35-as and C45-30-as, respectively. These two siRNAs still obeyed the general siRNA selection guidelines (29)
. All eight siRNAs were able to reduce target gene expression by 60–90% as measured by RT-PCR (Fig. 1G)
and immunoblotting (Fig. 1H)
experiments (data for some of the siRNAs not shown), whereas the transfection agent (Lane OF) and the control RNAs (Lane mm), with two to four mismatched bases, did not significantly affect the mRNA or protein levels of the target genes (Fig. 1, G and H)
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DNA Replication Is Inhibited by the Antisense ODNs and siRNAs That Can Silence the Target Genes.
Using BrdUrd incorporation assays, we showed that silencing of replication-initiation genes by antisense ODNs (Fig. 2, A and B)
and siRNA (Fig. 2C)
led to specific inhibition of DNA replication. For example, C6-35-as significantly reduced the number of HepG2 cells that incorporated BrdUrd, whereas the transfection agent alone and C6-35-mm had no effect (Fig. 2A)
. Similar results were also obtained for several other cell lines tested, as shown quantitatively in Fig. 2B
. Approximately 25–45% of untreated cells and of those treated with the transfection agent alone or C6-35-mm incorporated BrdUrd, indicating that these cells were in S phase during the labeling period, as expected for exponentially growing human cells in culture. In contrast, only 5% of cells were able to incorporate BrdUrd after transfection with C6-35-as in all cell lines tested (Fig. 2B)
. Therefore, C6-35-as inhibited DNA replication by 70–90% when the results were normalized to those for the untreated cells. Similar results were also obtained for other antisense ODNs tested, including M2-47-as, C45-18-as, and C45-30-as (data not shown). siRNAs inhibited DNA replication to degrees similar to those for the antisense ODNs (Fig. 2C)
. The results from both antisense ODNs and siRNAs are consistent with hCdc6p, hMcm2p, and hCdc45p being required for DNA replication in human cells.
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30–40% (Fig. 3A)
. This was consistent with our finding that although cell proliferation was impeded in both cancer and normal cells when DNA replication was inhibited, only cancer cells underwent apoptosis (see below). The residual viable cancer cells after the first transfection with the antisense ODNs were those that were un- or undertransfected cells because they could be inhibited to the same extent after regrowth, reseeding, and a second transfection by the same ODNs (data not shown).
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20% by the transfection agent alone and 30% by the mismatched control ODNs (Fig. 3A)
. These values were significantly lower than the 60–90% inhibition afforded by the antisense ODNs (Fig. 3A)
, consistent with sequence-specific inhibition of target gene expression by the antisense ODNs. The antisense ODNs also inhibited cell proliferation in other cancer cell lines: BEL-7402, T-Tn, NCI-H446, and NCI-H460 (data not shown). Six other antisense ODNs (C6-60-as, M2-61-as, M2-33-as, C6-33-as, M2-34-as, and C45-18-as) that could reduce expression of the target genes by 60% also inhibited cell proliferation by 60% (Fig. 1C)
. In addition, the activities of the antisense ODNs were dose dependent (see Fig. 3B
for an example), consistent with specific effects of the antisense ODNs in target gene silencing. Three siRNAs, siC6.1, siM2.1, and siC45.1, that were tested also inhibited proliferation of HeLa cells by 70–75% (data not shown).
Connection between Silencing of the Target Genes and Inhibition of Cell Proliferation.
As described above, selected antisense ODNs specifically inhibited target gene expression (Fig. 1, A–F)
, DNA replication (Fig. 2, A and B)
, and cell proliferation (Figs. 3A
and 1C
). We then asked whether the observed effects on cell proliferation and viability were the consequence of silencing of the target genes, as opposed to unintended cellular components, by the antisense ODNs. We first compared the inhibition of target gene expression with that of cell proliferation for all 16 antisense ODNs analyzed (Fig. 1C)
. A general trend was found: the greater the reduction of target gene expression (as reflected in lower amounts of residual mRNA), the more the inhibition of cell proliferation (as reflected in lower cell viability), suggesting that inhibition of cell proliferation was consequently linked to silencing of the target genes.
The above inference was supported by rescue experiments using a silently mutated hCdc6 gene whose sequence was changed in every wobble base on the target site for an antisense ODN, C6-35-as, without altering the encoded amino acids. Plasmid pC6SM (where SM denotes silent mutant), which expressed the silent mutant gene and was pretransfected into the cells, should have rescued the cells from the effects of C6-35-as if inhibition of cell proliferation resulted from silencing of the hCdc6 gene by C6-35-as. Similar rescue strategies have been used previously to examine specificities of antisense ODNs (30) and siRNAs (31) .
We first confirmed that hCdc6p was expressed from both HA-tagged and untagged versions of pC6SM and pC6WT (the wild-type hCdc6 gene) after transient transfection by immunoblotting with anti-HA and anti-hCdc6 antibodies (data not shown). After transfection with C6-35-as, the level of hCdc6p expressed from pC6WT, as well as that from the endogenous hCdc6p, was much reduced as expected, whereas that from pC6SM was only moderately reduced (data not shown). However, possible rescue of cell viability could not be easily observed by measuring the cell number of the entire cell population because the transfection efficiency for plasmid DNA (20%, as measured in a plasmid expressing GFP; data not shown) was much lower than that for ODNs. To overcome this problem, we cotransfected pC6SM with a plasmid expressing GFP (at a 4:1 molar ratio in favor of pC6SM or pC6WT) and monitored the percentage of GFP-positive cells in the population as the indication of cell viability after further transfection with C6-35-as. If the expected rescue occurred, the percentage of GFP-positive cells transfected with C6-35-as (normalized to cells not further treated) should have been higher for cells pretransfected with pC6SM than for those pretransfected with the vector control (pcDNA3.1) and those pretransfected with pC6WT.
As expected, pretransfection of the vector pcDNA3.1 did not rescue the cells from the effects of C6-35-as (Fig. 3C
; 18% GFP-positive cells, or 82% growth inhibition). On the other hand, pretransfection with pC6SM increased the number of GFP-positive cells to 45%, whereas pC6WT rescue was much lower, with 25% GFP-positive cells (Fig. 3C)
. The reason that pC6SM did not fully rescue the cells (in which case the GFP-positive cells would be 80%, as for the cells treated with the transfection agent instead of C6-35-as) is probably because some GFP-positive cells were under- or untransfected by pC6SM. As controls, the percentages of GFP-positive cells treated with the transfection agent alone or the mismatched control ODN (C6-35-mm) were not significantly affected by pretransfection with pC6SM, pC6WT, or pcDNA3.1 (Fig. 3C)
. These results strongly suggest that the inhibition of cell proliferation was the result of target gene silencing by the antisense ODN.
Apoptosis Occurs in Both p53-Positive and -Negative Cancer Cells But Not in Normal Cells When the Target Genes Are Silenced.
Not only was cell proliferation inhibited, but most cancer cells transfected with the antisense ODNs and siRNAs died, as observed under the microscope (data not shown). We then determined that the mode of cell death was apoptosis by several assays (Figs. 4
and 5)
. We first detected apoptosis by the TUNEL assay (32)
in HepG2 cells after transfection with C6-35-as but not with the transfection agent or C6-35-mm (Fig. 4A)
. Similar degrees of apoptosis were induced in all of the cancer cell lines tested (HepG2, Hep3B, HeLa, and Chang) by C6-35-as but not by the transfection agent or C6-35-mm, and normal cells (L-02) did not undergo apoptosis (Fig. 4, B and C)
. Similar results were obtained for other antisense ODNs analyzed, including M2-47-as, C45-18-as, and C45-30-as (data not shown). The siRNA siC6.1, but not the transfection agent alone or the mismatched RNA, also induced apoptosis in HeLa cells (Fig. 4D)
. We confirmed that similar levels of transfection (data not shown), of target gene silencing (Fig. 1)
, and of inhibition of DNA replication (Fig. 2)
were achieved in different cell lines by use of transfection conditions optimized for different cell lines. Therefore, because apoptosis occurred in both p53-wild-type (HepG2) and p53-defective (Hep3B) liver cancer cell lines, but not in normal liver cells (L-02), the results suggest that silencing of replication-initiation genes induces apoptosis in cancer, but not normal, cells and that the apoptosis is p53-independent.
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, middle row). Similar percentages of Annexin V-positive cells were observed for all cancer cell lines tested, whereas normal L-02 cells had few Annexin V-positive cells (Fig. 4F)
. Similar results (not shown) were obtained with all other antisense ODNs that were also tested in the TUNEL assays. Therefore, Annexin V staining assays support the conclusion drawn from the TUNEL assays. In addition, our data (Fig. 4E)
also suggest that cell death induced by the antisense ODNs was not necrosis because the cells were still positive for 6-CFDA, which measures the activity of cellular esterases (which convert 6-CFDA to 6-carboxyfluorescein; Ref. 34
) and should therefore be positive for early apoptotic cells, as well as for viable cells, but not for necrotic cells.
To determine whether apoptosis induced by the antisense ODNs proceeded via caspase activation, we added the caspase inhibitor Z-VAD to cell cultures immediately after transfection with the antisense ODNs and observed significantly reduced cell death as measured by the WST-1 assay (Fig. 4G)
and microscopic observations (data not shown). Without Z-VAD, the numbers of viable cells after transfection with the four antisense ODNs tested were 18–30% of the numbers of untreated cells, whereas Z-VAD increased the percentages to 40–65% (Fig. 4G)
. These results suggest that apoptosis induced by the antisense ODNs was caspase dependent.
Apoptosis of cancer cells was also observed by flow cytometric analysis of DNA content. HepG2 cells had a sub-G1 (<2C DNA) population (34.2%) after transfection with C6-35-as (Fig. 5A)
. On the other hand, most normal cells (L-02) showed G1 arrest (68.7%) with a reduction of the S and G2-M populations, and apoptosis was probably prevented as a consequence (Fig. 5B)
. As controls, the transfection agent and C6-35-mm did not significantly affect cell cycle distributions compared with the untreated cells in either the HepG2 or L-02 cell line (Fig. 5, A and B)
.
To examine the effects of inhibiting the expression of replication-initiation proteins in different phases of the cell cycle, we performed flow cytometry experiments with synchronized HeLa cells, which showed good synchrony with double thymidine block in G1 and subsequent release (Fig. 5C)
. Most cells had not entered the S phase 1 h after release, but most had completed the S phase by 6 h after release and then finished mitosis after 12 h. We selected the time points of 1 and 6 h after release from G1 block to transfect the cells with C6-35-as, using the transfection agent alone and C6-35-mm as the controls, and then analyzed the cells 8 h after transfection (with a transfection time of 4 h, the cells at harvest were at 12 h after release from G1). When the cells were treated with the transfection agent or C6-35-mm at 1 h after release (when the cells had not entered S phase, according to the data shown in Fig. 5C
), they were able to complete DNA replication and mitosis by the harvest time, as did the untreated cells (Fig. 5D)
, consistent with unimpeded cell cycle progression shown in Fig. 5C
. In contrast, the cells transfected with C6-35-as at 1 h after release were blocked in the S and G2-M phases (Fig. 5D
; 35.3% in S and 40.2% in G2-M phase), and apoptosis was observed at a later time point (20 h post-transfection; data not shown). On the other hand, when the cells were transfected with C6-35-as at 6 h after release from G1 (when they had completed DNA replication, according to the results shown in Fig. 5C
), they were able to undergo mitosis to become G1 cells by the harvest time, as did the untreated cells and those treated with the transfection agent or C6-35-mm (Fig. 5E)
. Therefore, our data (Fig. 5, C–E)
are consistent with the replication-initiation protein being required for DNA replication but not for mitosis, indicating that the antisense ODN did not have significant nonspecific effects on the cells, at least during mitosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data show that inhibition of cell proliferation and induction of apoptosis is consequently linked to silencing of the target genes by the antisense ODNs and siRNAs, as summarized below. (a) Several antisense ODNs and siRNAs largely silenced the corresponding target genes while leaving the expression of other functionally related replication-initiation proteins unaffected. (b) The 16 antisense ODNs targeted to three replication-initiation genes showed good agreement between their abilities to silence the target genes and to inhibit cell proliferation. (c) A silently mutated gene significantly lessened the effects of the antisense ODN, providing very strong evidence that the inhibition of cell proliferation and induction of apoptosis did not result from unintended inhibition of an unknown cellular target(s). (d) The antisense ODN did not block mitosis when synchronized cells were transfected after they had completed S phase, indicating that the antisense ODN did not cause any detectable harmful effect on cells in the G2-M phase. (e) Inhibition of the expression of the target genes by two different gene-silencing strategies, antisense and RNA interference, had similar effects on DNA replication, cell proliferation, and cell viability.
The p53-independent nature of apoptosis induced by inhibiting the expression of replication-initiation proteins is highly desirable for cancer therapy, because approximately half of all cancers are p53 deficient. Therefore, and because DNA replication is required for the growth of all cancers, this anticancer strategy should be broadly applicable. Furthermore, it is of high significance that normal cells, at least the L-02 cells that we examined, do not die when the expression of replication-initiation proteins is inhibited. This is probably because normal cells possess intact checkpoint controls to arrest the cell cycle in G1 phase to avoid cell death when DNA replication is prevented, whereas cancer cells usually lack normal checkpoint controls and will enter an abortive S phase, producing partially replicated chromosomes and resulting in chromosome damage and apoptosis.
Because the vast majority of human body cells are nonproliferating and thus do not express or need most replication-initiation proteins (1 , 2) ,3 antisense ODNs, siRNAs, and other agents that can silence these genes should not harm, or affect the cellular functions of, most normal cells in the body. This should provide the first level of selectivity of these agents as potential anticancer therapeutics. As for the small fraction of normal, proliferative body cells, they should still be able to perform their duties while arresting their cell cycles instead of entering into a fatal S phase when the expression of a replication-initiation protein is inhibited. This should provide another level of selectivity for cancer therapy: stopping proliferation of normal cells without interfering with their functions or killing them. We therefore suggest that inhibition of replication-initiation genes is an effective anticancer strategy that should not cause serious side effects.
It has been reported that gene silencing of Orc6p (a subunit of the initiator protein ORC, which is expressed in both stationary and cycling cells) by siRNAs results in deregulation of the cell cycle, including a block in mitosis and appearance of multinucleated cells (35) . It has also recently been shown that a fragment of the geminin protein, which inhibits the activity of the replication-initiation protein Cdt1p, can inhibit DNA replication and cell proliferation and lead to apoptosis of cancer cells in culture (36) . As proposed (36) , although geminin or a fragment thereof cannot be targeted to body cells, small molecules that may inhibit the activities of replication-initiation proteins are potential anticancer drugs. On the other hand, antisense ODNs (reviewed in Refs. 37, 38, 39, 40 ) and siRNAs (41, 42, 43) can inhibit gene expression and have been shown to exert expected biological actions in animals as well as in cultured cells. Therefore, antisense ODNs and siRNAs that can inhibit the expression of selective replication-initiation proteins may become effective anticancer agents. In addition, appropriate carriers (reviewed in Refs. 44 and 45 ) and/or chemical modifications may further increase the in vivo stability, delivery, cellular uptake, and thus efficacy of these potential anticancer drugs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Hong Kong Research Grants Council Grants DAG00/01.SC01 and HKUST6100/01M. 
2 To whom requests for reprints should be addressed, at Department of Biochemistry, HKUST, Clear Water Bay, Kowloon, Hong Kong. Phone: (852) 2358-7296; Fax: (852) 2358-1552; E-mail: bccliang{at}ust.hk
4 The abbreviations use are: ODN, oligodeoxynucleotide; siRNA, small interfering RNA; FBS, fetal bovine serum; WST-1, water-soluble tetrazolium {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio[-1,3-benzene disulfonate}. nt, nucleotide; as, antisense; mm, mismatched; GFP, green fluorescent protein; RT-PCR, reverse transcription-PCR; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling; 6-CFDA, 6-carboxyfluorescein diacetate.
Received 5/ 9/03. Revised 7/17/03. Accepted 8/26/03.
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