Materials and methods

Cells and cell culture

Human cervical cancer cell lines HeLa, C-33 A, and SiHa were purchased from the American Type Culture Collection (ATCC, Manassas, VA). SK-RC-29, a human renal cell carcinoma cell line, was generously provided by Dr Neil H Bander (New York Hospital-Cornell Medical Center, New York, NY).^{14, 23} 911, a transformed human embryonic retinoblast cell line, was obtained from Introgen (Leiden, Netherlands).²⁴ PER.C6, a transformed human embryonic retinoblast cell line, was obtained from DirectGene (Annapolis, MD).²⁵ HeLa, C-33 A and SiHa cells were cultured in minimum essential medium (MEM) (Gibco BRL, Grand Island, NY) and SK-RC-29 cells were cultured in RPMI 1640 (Gibco BRL). 911 and PER.C6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL). All media were supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin and 10% fetal bovine serum (FBS) and maintained at 37°C in 5% CO₂.

Construction and production of the replication-competent Ad-MN/CA9-E1a adenovirus

All plasmids were constructed according to the standard protocols.²⁶ Briefly, pMN/CA9E1a was constructed by inserting a 550-bp fragment of MN/CA9 promoter, which was cut out from pMN/CA9PGL3-P by the restriction enzyme EcoRI, into the unique EcoRI site in pBPAE1II. This pMN/CA9E1a shuttle vector was cotransfected with pJM17 into 911 cells using N-[1-(2, 3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) (Boehringer Mannheim, Indianapolis, IN) following the manufacturer's protocol²⁷ to generate the replication-competent adenovirus, Ad-MN/CA9-E1a. When 911 cells in the culture medium showed complete cytopathic effects, they were harvested and centrifuged at 1000 g for 10 minutes. The pooled supernatants were aliquoted and stored at -80°C as primary viral stock. Viral stocks were propagated in 911 cells and several clones of Ad-MN/CA9-E1a virus were selected by plaque purification. One of the viral clones was chosen, propagated in PER.C6 cells, harvested 36–40 hours after infection, pelleted, resuspended in phosphate-buffered-saline (PBS) and lysed. Cell debris was removed by centrifugation and viruses in the cell lysate were purified by CsCl gradient centrifugation. Concentrated viruses prepared above were dialyzed, aliquoted and stored at -80°C. The viral titer was determined by plaque-forming assay and optical density measurements.

cDNA production and PCR assay for MN/CA9 mRNA

Total RNA was extracted from tested cell lines by disrupting cells in Trizol solution (Gibco BRL). RNA was precipitated by ethanol, dissolved in water treated with diethylpyrocarbonate (DEPC) (Sigma, St Louis, MO). Complementary DNA (cDNA) for total RNA was synthesized by Moloney murine leukemia virus (M-MuLV) reverse transcriptase (MBI Fermentas, Hanover, MD) using random hexamer primers. In all, 2 g of total RNA and 0.2 g of random hexamer primers were mixed in 20 L reaction volume, placed at 70°C for 5 minutes and then placed on ice. The primer and RNA mixture were combined with 1 mM deoxynucleotide triphosphate, 20 U/L ribonuclease inhibitor, 50 mM Tris-HCl, 50 mM potassium chloride, 4 mM magnesium chloride and 10 mM DTT. The mixture was added to 20 U/L M-MuLV reverse transcriptase. The reverse transcription reaction was performed at 37°C for 1 hour. Samples were stored at -20°C.

PCR reaction was performed using MN/CA9 cDNA-specific primers following the methods described by Bartosova et al¹⁸ with minor modifications. Two primers (5'-CCG AGC GAC GCA GCC TTT GA-3' and 5'-TAG TCG ACT AGG CTC CAG TCT CGG CTA CTT-3') were used to amplify a particular 255-bp fragment within MN/CA9 cDNA. PCR was run for 35 cycles with denaturation at 95°C for 20 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 40 seconds. PCR products were analyzed on a 1% agarose gel. -Actin was also included for PCR amplification as an internal control.

Western blotting analysis for adenovirus E1a protein expression

For Ad5 E1a protein detection, 2 10⁶ cells plated in 100-mm dishes were infected with 1 MOI of Ad-MN/CA9-E1a for 14 hours. Cells were harvested and lysed in 200 L of cell lysis buffer (0.1 M Tris-HCl (pH 7.4), 0.5% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 M leupeptin) 2 days after viral infection. Lysates were centrifuged at 14,000 rpm for 20 minutes and the supernatants were collected. Total protein was estimated by dye binding assay (Bio-Rad, Hercules, CA). Protein (20 g) was loaded onto 12% SDS-PAGE gel. The proteins were transferred to polyvinylidene difluoride membrane (PVDF) (Scheleicher & Schuell, Dassel, Germany) and the membrane was probed with antibodies reactive to Ad5 E1a protein (MS-587-P1, NeoMarkers, Fremont, CA). Primary antibodies were detected using a goat anti-mouse secondary antibody conjugated to horseradish peroxidase and visualized by ECL (Amersham, Arlington Heights, IL).

In vitro cytotoxicity assay

HeLa, C-33 A, SiHa or SK-RC-29 cells were seeded onto 24-well plates at a density of 5 10³ cells/well. After 24 hours, cells were infected with 0.01–100 MOI of Ad-MN/CA9-E1a. Half of the medium was aspirated at days 3 and 5 after viruses were added (day 0) and replaced with fresh medium. The cell numbers per well were assessed at days 0, 1, 3, 5 and 7 by crystal violet assay. The data were presented as the percentage of cell numbers in the virus-infected group relative to the uninfected control group.

In vivo animal studies

Athymic BALB/c nude female mice, 6-week old, weighing 20 g (Japan SLC, Shizuoka, Japan) were inoculated subcutaneously with HeLa cells (2 10⁶ cells) in 100 L of 50% MATRIGEL basement membrane matrix (Becton Dickinson, Bedford, MA). When tumors became palpable (4–5 mm in diameter), animals were randomly assigned to two experimental groups, group 1 (n=7) for Ad-MN/CA9-E1a and group 2 (n=7) with PBS. A single dose of 50 L of Ad-MN/CA9-E1a (2 10⁹ pfu) or PBS was injected intratumorally with a microliter syringe fitted with a 28-gauge needle. Tumor volume was measured every 5 days and calculated using the following formula: volume (a rotational ellipsoid)=M₁ M₂² 0.5236 (M₁, long axis; M₂, short axis).²⁸ Tumor volumes were normalized to 100% at day 0. At 30 days after intratumoral injection, the mice were killed and tumors were removed for histological examination.

Results

RT-PCR analysis of MN/CA9 expression

Total RNA was prepared as described in Materials and methods from human cervical (HeLa, C-33 A and SiHa) and renal (SK-RC-29) cancer cells. HeLa cells are known to express MN/CA9 proteins^{8, 9} but SK-RC-29 cells do not.¹⁴ As expected, a distinct 255-bp fragment corresponding to the sequence within MN/CA9 cDNA was detected in all three human cervical cancer cells tested, but SK-RC-29 cells did not have MN/CA9 transcripts. HeLa cells showed the most abundant expression of MN/CA9 (Fig 1).

Figure 1.

RT-PCR assay identifies the 255-bp fragment in MN/CA9 cDNA in HeLa, C-33 A and SiHa cell lines, but not in the SK-RC-29 cell line. -Actin was included to confirm RNA preparation.

Full figure and legend (12K)

Western blotting analysis of adenovirus E1a protein expression

Ad5 E1a protein production in HeLa, C-33 A, SiHa or SK-RC-29 cells after infection with Ad-MN/CA9-E1a viruses was determined by Western blotting (Fig 2). E1a proteins were not detected in any cells prior to infection (data not shown). After infection, MN/CA9-positive cells (HeLa, C-33 A and SiHa) synthesized several E1a proteins with a molecular weight of 35–46 kDa. However, MN/CA9-negative cells (SK-RC-29) revealed a much lower level of E1a expression. As expected from RT-PCR results, MN/CA9 promoter mediated sufficient amounts of E1a expression to support the viral replication in MN/CA9-positive cells, but not in MN/CA9-negative cells.

Figure 2.

Expression of Ad5 E1a protein by Ad-MN/CA9-E1a was evaluated in different cell lines. A large amount of E1a proteins ranging in size from approximately 35–46 kDa were detected in HeLa, C-33 A and SiHa cervical cancer cells. However, SK-RC-29 cells had a much lower level of E1a proteins.

Full figure and legend (19K)

Selective cytotoxicity of Ad-MN/CA9-E1a to MN/CA9-positive cell lines in vitro

To evaluate the selective cytotoxicity of Ad-MN/CA9-E1a virus, we infected each cell line in vitro with a wide range (0.01–100 MOI) of viruses (Fig 3). The growth of MN/CA9-positive cells was significantly inhibited by small virus particles of Ad-MN/CA9-E1a: HeLa, 0.1 MOI; C-33 A, 0.01 MOI; SiHa, 1 MOI. Unlike MN/CA9-positive cell lines, the growth of the MN/CA9-negative cell line SK-RC-29 was not affected by 0.01–1 MOI. As many as 100 MOI were required to inhibit its growth.

Figure 3.

Ad-MN/CA9-E1a induced the selective growth inhibition of MN/CA9-positive cancer cells. After cells were exposed to 0.01–100 MOI of Ad-MN/CA9-E1a, cell growth was evaluated in vitro. Data are expressed as the percentage of cell numbers in the Ad-MN/CA9-E1a-infected group relative to the uninfected control group. Unlike the growth of MN/CA9-negative SK-RC-29 cells (d), growth of MN/CA9-positive cells, HeLa (a), C-33 A (b), and SiHa (c), was significantly inhibited by a small number of viruses. Results are represented as meanSD.

Full figure and legend (31K)

In vivo growth inhibition of HeLa xenograft by Ad-MN/CA9-E1a virus

Human cervical HeLa tumors were induced by subcutaneous injection of HeLa cells into athymic nude mice. After tumor formation, animals were intratumorally injected with Ad-MN/CA9-E1a or PBS as a negative control. The time of virus injection was considered day 0. Tumor volumes were measured at the times indicated in Figure 4. Ad-MN/CA9-E1a viruses effectively caused growth delay of HeLa xenografts. After 25 days, two of seven mice were visually free of tumor (Fig 5). Light microscopic observation of tumors in mice injected with Ad-MN/CA9-E1a showed that almost all cancer cells underwent necrosis (Fig 6). However, tumors in mice injected with PBS showed that cancer cells grew well.

Figure 4.

Xenografts were treated with Ad-MN/CA9-E1a. HeLa tumor xenografts were grown s.c. in athymic nude mice. Tumors were treated with Ad-MN/CA9-E1a (n=7) or PBS (n=7) by intratumoral injection at day 0 and measured every 5 days. Tumor volumes were normalized to 100% at day 0. The data are represented as meanSD.

Full figure and legend (11K)

Figure 5.

After intratumoral injection of Ad-MN/CA9-E1a, the tumor growth of the subcutaneous HeLa tumor xenografts was inhibited at 30 days. (a) Tumor has regressed in mice treated with Ad-MN/CA9-E1a. Two of the seven mice with Ad-MN/CA9-E1a intratumoral injection were visually free of tumor. (b) Big tumors were still seen in mice treated with PBS.

Full figure and legend (50K)

Figure 6.

Tumors were removed for histological examination after the mice were killed at 30 days. (a) Nearly all cancer cells were necrotized in mice treated with Ad-MN/CA9-E1a (H&E, 200). (b) Cancer cells were growing well in mice treated with PBS (H&E, 200).

Full figure and legend (59K)

Discussion

Replication-defective recombinant adenoviruses have been widely studied in vivo and in vitro as a vector to deliver cancer therapeutic genes. They can transduce a variety of mammalian cells in a cell replication-independent manner. However, there are several limitations in the use of these vectors for cancer gene therapy. These vectors do not infect all of the cancer cells and permit only a short duration of exogenous gene expression. To circumvent these limitations, replication-competent oncolytic viruses have been developed as an alternative strategy for cancer gene therapy. These can replicate and kill infected cells by viral lysis. Furthermore, the input viral dose is amplified by replication, spreading to adjacent cancer cells after lysis of initially infected cells.^{19, 20, 21, 22}

Conditional viral gene expression and subsequent replication were achieved using a tissue-specific promoter, resulting in selective replication in the tumor without the damage to neighboring normal cells.^{19, 20, 21, 22} There have been several reports comparing viral promoters with the tissue-specific promoters. The minimal enhancer/promoter from the prostate-specific antigen (PSA) gene has been used to drive E1a expression and create an adenovirus, CN706, that selectively replicates in PSA-positive prostate cancer cells.²⁹ The alpha-fetoprotein (AFP) promoter has also been used to develop the therapeutic adenovirus AvE1a04i, which selectively replicates in AFP-expressing hepatocellular carcinoma cells.³⁰

MN/CA9 is a transmembrane glycoprotein with apparent molecular weights of 58 and 54 kDa, which was first identified in HeLa cells.^{8, 9, 10} Recent reports confirm that MN/CA9 is a potentially important biomarker for uterine cervical carcinoma. MN/CA9 protein was expressed in more than 90% of uterine cervical cancer tissues, but normal cervical tissues did not express this antigen.¹¹ There was also an association between MN/CA9 immunostaining of atypical cells in Papanicolaou smears and the presence of significant lesions (adenocarcinoma in situ/carcinoma and cervical intraepithelial neoplasia II or III) in the cervix.¹² MN/CA9 expression was upregulated in hypoxic cervical cancers and was associated with a poor prognosis in locally advanced uterine cervical cancers.³¹ Although the functional significance of MN/CA9 in oncogenesis remains unclear, some evidence supports the role of the MN/CA9 protein in neoplastic progression. MN/CA9 induced a malignant phenotype in murine NIH 3T3 fibroblasts following transfection with MN/CA9 cDNA.¹⁰ Also, MN/CA9 expression correlated with the tumorigenic phenotype of HeLa fibroblast somatic cell hybrids; MN/CA9 protein was absent in nontumorigenic hybrid cells (clone CGL1), but it was found in tumorigenic segregants (clones CGL3 and CGL4). Therefore, MN/CA9 may be involved in the control of cell proliferation and transformation.^{10, 32, 33, 34} Based on this evidence, MN/CA9 promoter was used to develop an adenovirus, Ad-MN/CA9-E1a, which selectively replicates in MN/CA9-expressing uterine cervical cancer cells. The goal of this study was to create a MN/CA9 promoter-directed replication-competent adenovirus that could display tumor-specific replication competency and be directly cytotoxic only to uterine cervical cancer cells.

To date, 293 cells generated by transformation of human embryonic kidney cells by sheared Ad5 DNA are the most widely used helper cell line for the production of recombinant adenovirus.³⁵ However, we chose 911 and PER.C6 cell lines in this experiment because these cell lines possess several favorable characteristics compared to 293 cells. The 911 cell line is an Ad5 E1-transformed human embryonic retinoblast cell with a plasmid containing base pairs 79–5789 of the Ad5 genome. This cell line has comparable quality to 293 cells with respect to transfection efficiency and the frequency of homologous recombination. It also exhibits a shortened plaque-forming time in plaque assay and enhanced viral yields in small-scale production of adenoviral vectors in comparison with 293 cells.²⁴ Most importantly, since Fallaux et al²⁴ observed that replication-competent adenoviruses were not generated in the 911-produced replication-defective adenoviral vector batches, we used this cell line for making our replication-competent adenovirus, Ad-MN/CA9-E1a. However, 2 years later, the same group reported that 911 cells could also generate a replication-competent adenovirus in replication-defective adenoviral vector batches and developed a new helper cell line, PER.C6. The PER.C6 cell line, which contains the Ad5 E1a- and E1b-encoding sequences (Ad5 nucleotides 459–3510) under the control of the human phosphoglycerate kinase (PGK) promoter, does not result in the generation of replication-competent adenoviruses.²⁵ Therefore, we used the PER.C6 cell line for the amplification of Ad-MN/CA9-E1a.

The tissue-specific replication and cytotoxicity of Ad-MN/CA9-E1a adenovirus was assessed in four different cancer cell lines. All three of these uterine cervical cancer cell lines synthesized MN/CA9. HeLa cells, which are known to express MN/CA9⁸, showed the strongest expression of MN/CA9 as assessed by RT-PCR assay. Since one of the renal cell carcinoma cell lines, SK-RC-29, is known to be MN/CA9 negative,¹⁴ we included this cell line as a negative control, confirming the negligible level of MN/CA9 transcripts. Coinciding with the results of RT-PCR assay, Western blotting showed the tissue-specific promoter activity enabling the selective E1a expression in MN/CA9-expressing cells. The in vitro tissue-specific cytotoxicity of Ad-MN/CA9-E1a in MN/CA9-expressing cells was assessed. Whereas the growth of MN/CA9-positive uterine cervical cancer cell lines was significantly inhibited by a small number of Ad-MN/CA9-E1a virus particles, the growth of the MN/CA9-negative cell line SK-RC-29 could only be inhibited by a much greater exposure to Ad-MN/CA9-E1a. X-gal staining after infection with Ad-RSV-gal in SK-RC-29 cells showed that the percentages of -gal transduced cells were 34% at 2.5 MOI, 45% at 5 MOI, 72% at 10 MOI and 91% at 20 MOI.³⁶ This suggests that SK-RC-29 cells have a good viral infectivity and the differential cytotoxic effects of Ad-MN/CA9-E1a is due to the difference in MN/CA9 transcriptional activity of the cells, but not that in viral infectivity. Collectively, these results demonstrate that Ad-MN/CA9-E1a has selective cytotoxicity in MN/CA9-expressing cells with a good therapeutic window. As expected, the intratumoral administration of Ad-MN/CA9-E1a effectively inhibited the growth of HeLa xenografts in athymic nude mice. After 25 days of viral injection, two of the seven mice were visually free of tumor.

In conclusion, we have established a novel replication-competent adenoviral vector using MN/CA9 promoter to drive the replication of adenovirus only in MN/CA9-expressing cancer cells, with selective cytotoxicity and a good therapeutic window. This novel vector system may have potential as a strategy for the treatment of uterine cervical cancer.

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Acknowledgements

We thank Dr Hee Jae Joo, Department of Pathology, for assistance in the preparation and interpretation of histologic studies. This work was supported by 2001 grant from the Department of Medical Sciences, The Graduate School, Ajou University.