Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1998 Nov;72(11):9201–9207. doi: 10.1128/jvi.72.11.9201-9207.1998

Combination Gene Delivery of the Cell Cycle Inhibitor p27 with Thymidine Kinase Enhances Prodrug Cytotoxicity

Xavier Danthinne 1,†, Kazunori Aoki 1,2, Akiko L Kurachi 1, Gary J Nabel 1,2,3, Elizabeth G Nabel 1,*
PMCID: PMC110339  PMID: 9765467

Abstract

Cytoxicity induced by the herpesvirus thymidine kinase (TK) gene in combination with prodrugs is dependent on cell growth and leads to the elimination of genetically modified cells, thus limiting the duration of expression and efficacy of this treatment in vivo. Here, an effort was made to enhance TK/prodrug efficacy by coexpression of a cyclin-dependent kinase inhibitor (CKI), p27, to render cells resistant to TK/prodrug by inhibiting DNA synthesis. Expression of p27 by transfection substantially reduced cell cycle progression, and its activity was enhanced by mutations designed to stabilize the protein. Coexpression of p27 and TK or a p27/TK fusion protein led to greater prodrug cytotoxicity than that produced by TK alone in the Renca cell line, which is sensitive to bystander killing. Combination gene transfer of this CKI with TK therefore sustained the synthesis of TK by genetically modified cells to enhance the susceptibility of bystander cells to prodrug cytotoxicity and increased the efficacy of this gene transfer approach.

The thymidine kinase (TK) gene from herpes simplex virus type 1 (HSV-1) is widely used as a cytotoxic gene in combination with prodrugs in different mammalian gene transfer and transgenic systems. HSV TK phosphorylates the guanosine analogs ganciclovir (GCV) and acyclovir (ACV) more efficiently than cellular TK does, and the monophosphate drugs are subsequently phosphorylated by cellular enzymes into their triphosphate forms (3), which are incorporated into elongating DNA, leading to elongation arrest (ACV) or decreased DNA synthesis (GCV) (4, 5, 14, 17, 21, 39). Death usually ensues, through a mechanism identified in some cases as apoptosis (7, 35), although the mechanism and pathways that lead to cell death are not completely understood.

One feature of this gene transfer/prodrug approach is the generation of bystander cytotoxicity that leads to the death of untransduced cells adjacent to genetically modified cells. Several potential mechanisms have been proposed to mediate this phenomenon. Freeman et al. hypothesized that the uptake of phosphorylated GCV by bystander cells occurs via the endocytosis of apoptotic vesicles, originating from the TK-transduced cells and containing the toxic drug (12); however, increasing evidence suggests that the bystander effect is mediated via gap junctions that allow phosphorylated ganciclovir to translocate from TK+ to TK− cells intercellularly (2, 11). Although the bystander effect can be observed in vitro, an immune component might be involved in some tumor models since this phenomenon is impaired or even absent in immunocompromised animals (7, 13).

The TK/GCV system has been successfully applied in cancer and cardiovascular models in vivo (8, 10, 21, 22, 25, 29); however, the efficiency of gene delivery in vivo remains low. Because of their potential antitumor activity, cytokines have been combined with TK. Ram et al. constructed retroviral vectors carrying both the HSV TK and interleukin-2 (IL-2) genes, but no enhancement of tumor eradication was observed upon transduction of rat 9L gliosarcoma (31). Cotreatment of established tumors with TK- and IL-2-expressing adenoviral vectors was shown to enhance eradication of metastatic colon carcinoma in mouse liver (6) and head and neck cancer in mice (26, 27). In nude mice, coinjection of C6 glioma cells with retroviral producer cells expressing TK and IL-4 appeared to inhibit tumor growth more effectively than coinjection with cells expressing TK only (1). In another approach, Rogulski et al. fused the sequences encoding TK and Escherichia coli cytosine deaminase and observed a slight synergistic toxicity and an enhanced radiosensitivity in glioma cells (33).

In this study, we have explored an alternative strategy to increase cell killing by TK/GCV. Since administration of GCV in the presence of HSV TK leads to lysis caused by its effects on DNA replication, we hypothesized that the growth arrest of gene-modified cells would render them less sensitive to TK/GCV-mediated killing and prolong the duration of TK expression, thereby sustaining local conversion of GCV and the cytotoxic effect on adjacent cells. We have found that combination gene transfer of TK and a cyclin-dependent kinase inhibitor (CKI) enhanced bystander cell killing in the presence of GCV.

MATERIALS AND METHODS

Plasmids.

The cDNAs which encode human p21 (42), p16 (37), p27 (30), HSV-1 TK, human alkaline phosphatase (hAP) (24), and human CD2 were inserted in VR1012, a eukaryotic expression vector that contains a cytomegalovirus (CMV) immediate-early gene promoter, enhancer, and intron and a bovine growth hormone polyadenylation signal. A plasmid expressing human immunodeficiency virus type 1 (HIV-1) Vpr under control of the CMV immediate-early gene promoter and a simian virus 40 polyadenylation signal was a gift from E. Cohen (University of Montreal, Montreal, Canada).

A bicistronic construct expressing p27 and TK (pCMVp27citeTK) was made by insertion of the EcoRI-NcoI fragment from pCITE-1 (Novagen, Madison, Wis.) between an XbaI site located immediately downstream from the p27 coding sequence and an NcoI site containing the initiator codon of the TK gene. This EcoRI-NcoI fragment (“CITE”) contains a copy of the encephalomyocarditis virus RNA 5′ noncoding region, which functions as an internal entry point for initiation of translation by eukaryotic ribosomes. As a control for p27 activity, a vector containing the p27 coding region but in reverse orientation with respect to the CMV promoter, pCMVp27revcite TK, was prepared similarly. To reduce the size of the expression cassette, a SacII-EcoRV fragment containing the CMV intron was deleted in both vectors.

Mutation of the Cdc2 kinase consensus phosphorylation site on p27 from TPKK to AAGG was performed by using overlapping PCR-based methods with plasmid pCMVp27citeTK as a template. On one side, sequences corresponding to nucleotides 186 to 576 from the start of the p27 coding region were amplified by using the oligonucleotides 26 (5′-CGATTTTCAGAATCACAAACCCC-3′) and 24 (5′-GCCAGGCCCCCCGGCCGCCTGCTCCACAGAACC-3′) as primers. On the other side, sequences corresponding to position 554 from the start of the p27 coding region to the BglI site located in the downstream CITE sequences were amplified by using the oligonucleotides 23 (5′-GAGCAGGCGGCCGGGGGGCCTGGCCTCAGAAG-3′) and 27 (5′-TTTGGCCGCAGAGGCACCTGT-3′). Mutations in oligonucleotides 23 and 24 are indicated in boldface type. Both PCR products were amplified in a single reaction by using oligonucleotides 26 and 27 as primers, with 6 cycles (94°C, 15 s; 45°C, 30 s; 72°C, 45 s) followed by 30 cycles (94°C, 15 s; 65°C, 30 s; 72°C, 45 s). The resulting DNA fragment was digested with SacII and XbaI and inserted into pCMVp27citeTK to replace the corresponding fragment. The integrity of the sequences was verified by sequencing.

A fusion protein between p27 and TK was made by deleting an AatII-NcoI fragment from pCMVp27citeTK, giving rise to plasmid pCMVp27TK. The resulting protein had the last four amino acids of p27 (RRQT) deleted, and an additional serine residue was inserted in front of the first methionine residue of the TK. A fusion between the NH2-terminal part of the p27 coding region and the sequences encoding the TK was created by deleting the SacII-NcoI fragment from pCMVp27citeTK, giving rise to plasmid pCMVp27SNTK. Similarly, the SacII-FspI and MarI-FspI fragments were prepared by deletion of this fragment from pCMVp27TK, pCMVp27SFTK, and pCMVp27NFTK, respectively. The open reading frame between the SacII and FspI sites was maintained by inserting complementary oligonucleotides (5′-GGTCGAC-3′ and 5′-GTCGACCGC-3′). Introduction of the NH2-terminal part of p27 downstream of the cyclin-CDK2 binding domain of p27 was performed by ligating a NcoI-HindIII fragment from plasmid VR1012-p21N between the SacII and FspI sites of pCMVp27TK, giving rise to plasmid pCMVp27Sp21FTK. VR1012/p21N contains a copy of the sequences coding for the first 75 amino acids of p21. Similarly, pCMVp27Np21FTK was constructed by inserting the same NcoI-HindIII fragment between the NarI and FspI sites of pCMVp27TK.

293 cell transfections and fluorescence-activated cell sorter (FACS) analysis.

293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum at 37°C and 5% CO2. Cells (2 × 106) inoculated the previous day in 10-cm-diameter culture dishes were transfected with 15 μg of plasmid DNA by using CaPO4 transfection. For cell cycle analysis, 293 cells were typically transfected with 3 μg of a CD2 expression plasmid and 12 μg of the relevant CKI expression plasmid.

One day after transfection, cells were detached from the tissue culture dish with phosphate-buffered saline (PBS) containing 2 mM EDTA. Cell clusters were disrupted by pipetting, and 106 cells were plated in a 15-cm diameter dish. The next day, the cells were harvested, and the CD2 cells were analyzed for DNA content by flow cytometry as previously described (36). Briefly, 106 cells were incubated with 50 μl of anti-CD2 mouse hydridoma supernatant (ATCC HB222) for 20 min on ice. The cells were washed twice with 1 ml of PBS–2% fetal calf serum and incubated with 0.2 μg of fluorescein isothiocyanate-conjugated sheep anti-mouse immunoglobulin in 50 μl of PBS–2% fetal calf serum for 20 min on ice. The cells were washed with 1 ml of PBS–2% fetal calf serum and fixed in 0.25% paraformaldehyde–PBS for 1 h on ice. The fixed cells were permeabilized with 0.2% Tween 20–PBS for 15 min at 37°C. The cells were washed again with 1 ml of PBS–2% fetal calf serum and incubated for 1 h at 37°C in 1 ml of PBS containing 30 μg of propidium iodide and 2 U of DNase-free RNase (Boehringer Mannheim) per ml. Fluorescence was analyzed on a FACScan (Becton Dickinson) flow cytometer. Data represent at least 10,000 events corresponding to the cells expressing the highest CD2 levels. The DNA profiles were analyzed by using Modfit LT software (Verity Software House, Inc.).

Assays of proliferation and bystander effect.

Renca cells were maintained in RPMI medium supplemented with 10% fetal calf serum at 37°C and 5% CO2 in 10-cm-diameter dishes until they reached 90% confluence; they were then transfected with 25 μg of DNA complexed with 100 μg of Lipofectamine (Gibco BRL). For bystander experiments, Renca cells were typically transfected with 5 μg of CD2 and 20 μg of TK expression vector plasmids.

One day after transfection, cells were harvested and diluted with increasing amounts of untransfected cells. A total of 104 cells were plated per well in a 96-well microtiter plate and incubated for 6 h at 37°C to permit cell adherence to the plate. The medium was then changed to fresh medium containing 5 μM GCV. Cultures were terminated at 5 days, and cell proliferation was measured by using a colorimetric cell proliferation assay (23). To determine transfection efficiencies, 0.5 × 106 cells in a 10-cm-diameter culture dish were incubated for 2 days at 37°C, harvested, and analyzed by FACS for CD2 expression as described above.

RESULTS

Transfection of p27 plasmid induces growth arrest more effectively than p21, p16, or Vpr.

Because inhibition of DNA synthesis by TK/GCV has been implicated in cell death, we postulated that arrest of cell cycle progression may render transfected cells less sensitive to TK/GCV and prolong TK expression to enhance potency. Inhibition of cell cycle progression can be achieved by expression of several gene products, including the p16, p21, and p27 CKIs, which arrest cell cycle progression in G0 or at the G1/S boundary. HIV-1 Vpr inhibits the activity of cyclin B-Cdc2 and arrests the cell cycle at the G2/M checkpoint (9).

The first step was to identify the most potent CKI that could be combined subsequently with TK. CKIs were expressed under the control of the same CMV enhancer-promoter and were cotransfected with a plasmid expression vector encoding the cell surface marker CD2 (pCMV-CD2) into a highly transfectable cell line, 293 (15), or into Renca cells, a murine epithelial carcinoma cell line. Cells which expressed CD2 as a marker were analyzed for their DNA content by flow cytometry. In p21- and p27-transfected 293 cells, the proportions of cells in G1 were 58 and 76%, respectively, in comparison to 27% for the control (Fig. 1). p16 did not show activity in 293 cells, although it was readily detected by Western blot analysis (data not shown) and inhibited Cdk4 kinase activity (40). Expression of Vpr led to an accumulation of about 42% of the cells in the G2 phase. In p21-, p27-, and p16-transfected Renca cells, the percentages of the CD2-positive cells in G1 were 66, 76, and 63%, respectively, in comparison to 44% for the control. Interestingly, Vpr did not show any activity in Renca cells. Because p27 showed the greatest reduction in cell cycle progression in both cell lines, it was examined further for its effect on TK/GCV cytotoxicity in cotransfected cells.

FIG. 1.

FIG. 1

Open in a new tab

Overexpression of p27 arrests the cell cycle more strongly than overexpression of p21, p16, or Vpr. A vector expressing p21, p27, p16, Vpr, or human alkaline phosphatase (hAP) under the control of a CMV enhancer-promoter was transfected into 293 or Renca cells together with a CD2-expressing plasmid. After 2 days of expression, the cells were harvested and stained simultaneously with an anti-CD2 antibody and propidium iodide. The histograms represent the DNA profiles of the cells expressing the highest CD2 levels. The fraction of cells in each phase of the cell cycle is indicated above the corresponding peak. In 293 cells, the percentages of CD2-positive cells were 62, 60, 67, 60 and 63% for panels from top to bottom, respectively. In Renca cells, these percentages were 5.7, 6.8, 6.2, 7.8, and 7.4%, respectively.

A vector which coexpressed p27 and TK was prepared by inserting both coding sequences in a single transcription unit, with p27 inserted downstream from the CMV promoter, followed by a cis-acting internal translational entry site (cite) and the HSV-1 TK gene, giving rise to pCMVp27citeTK. As a negative control, the p27 coding sequence was inserted in antisense orientation to generate pCMVp27revciteTK. The ability of these vectors to induce G1 growth arrest in 293 cells was tested by cotransfection of pCMVp27citeTK and pCMV-CD2. Two days after transfection, CD2+ cells were analyzed for DNA content. pCMVp27citeTK was comparable to pCMVp27 in its ability to cause G1/S growth arrest, with 55 and 51% of cells in G1, respectively (Fig. 2A and B). The effect of TK on cell growth was measured in the presence or absence of GCV in these expression vector plasmids. Cells transfected with the vectors expressing TK did not proliferate for up to 6 days after transfection in the presence of GCV (Fig. 2C). Comparable levels of TK expression were observed from the pCMVTK and pCMVp27citeTK plasmids by Western blot analysis (Fig. 2D), indicating that the differences in their activity are due to the expression of p27. In addition, because 293 cells do not show significant bystander killing and were nearly completely transfected (data not shown), this result suggested that most cells expressed sufficient TK to be lysed in the presence of GCV. Taken together, these results suggest that p27 and p27/TK expression vectors were comparably effective in arresting cell cycle progression and that TK remained functional in this vector. To confirm that cells which express p27 and TK were more viable in the presence of GCV than those with TK alone, 293 cells were transfected with a set of p27/TK expression plasmids and a CD2 expression plasmid. GCV was added 1 day after transfection, and the cells were harvested 4 days later. The percentage of CD2+ cells was determined by FACS analysis. Twenty to 22% of the strongly positive CD2 cells transfected with pCMVTKcitep27 or pCMVp27citeTK were detected, compared to ≤12% for the cells transfected with pCMVTKcitep27rev or pCMVp27revciteTK. The cells expressing the highest levels of CD2 were also most efficiently arrested by p27 (when CD2 expression was increased, G1 growth arrest was higher). These results suggested that cells arrested by p27 survive GCV treatment better than growing cells. Before testing the efficacy of combination gene transfer in bystander killing, the growth arrest activity of p27 was optimized further.

FIG. 2.

FIG. 2

Open in a new tab

Coexpression of p27 and TK in 293 cells. (A) Schematic representation of the constructs expressing p27 and TK. (B) DNA profiles of 293 cells transfected with the respective plasmids illustrated in panel A and a CD2-expressing plasmid. All of the CD2-expressing cells were included in the analysis. The fraction of cells in G1 phase of the cell cycle is indicated above the corresponding peak. The fractions of CD2-positive cells for each transfection (from left to right) were 65, 61, 68 and 64%, respectively. (C) Growth of 293 cells transfected with the respective plasmids illustrated in panel A in the presence or in the absence of 5 μM GCV. Proliferation was measured by using a colorimetric assay. Data represent the average of three measurements. OD570–650, optical density at 570 to 650 nm. (D) Comparable expression of TK in different pCMVTK (lanes 1 and 2) and pCMVp27citeTK (lanes 3 and 4) plasmid expression vectors. Western blot analysis was performed by standard methods (40) with a polyclonal rabbit antiserum to HSV TK, kindly provided by William Summers (Yale University).

Optimization of G1/S growth arrest by p27 expression vectors.

The structure of p27 includes an NH2-terminal region similar to p21 and contains a CDK binding region (amino acids 28 to 79) (30, 41). Expression of this domain of p27 is sufficient to inactivate cyclin A- or E-Cdk2 complexes and cause G1/S growth arrest. The function of the COOH-terminal region of p27 has not been established, although it binds to E1A and contains a putative nuclear localization signal (amino acids 153 to 169) and a consensus Cdc2 phosphorylation site (amino acid T187).

To increase p27 activity, the consensus Cdc2 phosphorylation site TPKK was mutated to AAGG in pCMVp27citeTK, giving rise to pCMVp27ΔcdcciteTK. Indeed, it was recently shown that cyclin E-Cdk2 phosphorylates p27 on T187, promoting the degradation of p27 and subsequent transit from G1 to S phase (38). Expression of this vector resulted in more cells arrested in G1 than expression of wild-type p27 did (Fig. 3). An alternative p27 mutant was made by fusion of the COOH terminus of p27 to the NH2 terminus of TK (pCMVp27TK). This fusion gene product provided the advantage of a single open reading frame which would allow its combination with a third gene that might further enhance the action of p27 and TK. Transfection of 293 cells with the p27/TK fusion protein plasmid showed that it was comparably active to p27 in the bicistronic vector (Fig. 4). In the absence of GCV, cells transfected with pCMVp27TK, pCMVp27citeTK, or pCMVp27 had similar growth curves. In the presence of GCV, cell proliferation was strongly inhibited in cells transfected with pCMVp27citeTK and pCMVp27revcite TK and to a lesser extent with pCMVp27TK. Results obtained with a colorimetric proliferation assay were comparable to those observed by determination of cell numbers (Fig. 4B and C).

FIG. 3.

FIG. 3

Open in a new tab

Effect of Cdc2 consensus site mutation on the activity of p27. DNA profiles of 293 cells transfected with plasmid pCMVp27revciteTK (A), pCMVp27citeTK (B), or pCMVp27cdcciteTK (C) together with pCMV-CD2. All of the CD2-expressing cells were included in the analysis. The percentages of these transfected cells in the entire cell population were 82, 81 and 74%, respectively. The fraction of cells in each phase of the cell cycle is indicated above the corresponding peak.

FIG. 4.

FIG. 4

Open in a new tab

A p27-TK fusion protein which retains both activities. 293 cells were transfected with plasmids pCMVp27TK (fusion) (⧫), pCMVp27citeTK (■), pCMVp27revciteTK (▴), pCMVp27 (▵), and pCMVp27rev (•). One day after transfection, cells were seeded in a 96-well plate and cultured in the absence (A) or presence (B and C) of 5 μM GCV. Cell proliferation was measured by use of a colorimetric assay (A and B) or by counting viable cells (trypan blue staining) (C). OD570–650, optical density at 570 to 650 nm.

Since the activity of p27 is regulated by protein degradation via the ubiquitin-proteasome pathway (28), we reasoned that deletion of the recognition sequence for the ubiquitination apparatus, similar to mutations of the destruction boxes of cyclin B (18), would increase p27 stability and activity. Internal deletions were made in the COOH-terminal region of p27, leaving the NH2-terminal region which binds to CDK intact. Analysis of these mutants revealed that they induced G1/S growth arrest more effectively than wild-type p27 did (Fig. 5). Eighty-two to 85% of cells were found in G1 when the sequences between NarI and FspI sites or SacII and FspI sites were deleted, in comparison to 73% for the complete protein or 77% with a protein with the COOH-terminal region completely deleted. Thus, the activity of p27 could be increased by deletion of regions implicated in the degradation of this protein and was also retained in the p27/TK fusion protein which retained both activities at levels comparable to each individual wild-type protein.

FIG. 5.

FIG. 5

Open in a new tab

Effect of deletions in the p27 coding region on p27 activity. 293 cells were transfected with plasmids pCMVp27TK (A), pCMVp27NFTK (B), pCMVp27SFTK (C), pCMVp27SNTK (D), and pCMVTK (E), together with pCMV-CD2. The transfection efficiencies were 65, 71, 68, 68, and 67% CD2+ cells, respectively. The histograms represent the distribution of the CD2+ cells throughout the cell cycle 2 days after transfection.

Combination p27 and TK gene transfer enhances bystander cell killing.

To analyze its efficacy in prodrug-mediated killing, we transfected Renca cells, a murine epithelial carcinoma cell line, with pCMVp27citeTK, pCMVp27TK, and pCMVp27-revciteTK, together with pCMV-CD2, which served as a control reporter to standardize transfection efficiency. One day after transfection, transfection efficiencies were determined, and cells were harvested and mixed with increasing numbers of nontransfected cells. GCV was added to the tissue culture medium, and cell proliferation was analyzed 4 days later. GCV-induced killing was undetectable in cells cocultured with ∼8% pCMVp27revciteTK-transfected cells, probably reflecting the replacement of dead cells by the fast-growing untransfected cells. In contrast, cell death was readily observed at these ratios for the other expression vectors, with the pCMVp27TK fusion plasmid being more potent than pCMVp27citeTK (Fig. 6). Since a minority of cells were transfected but each vector had a similar TK activity in 293 cells (Fig. 4), it is likely that this difference is due to the bystander effect previously documented to occur in Renca cells (25).

FIG. 6.

FIG. 6

Open in a new tab

Coexpression of p27 with TK enhances cell killing in a murine sarcoma cell line. Renca cells were transfected with the plasmids indicated above the graphs, together with pCMV-CD2. The transfection efficiencies (left to right) were 8.0, 8.2, and 7.7% CD2+ cells, respectively. One day after transfection, the cells were mixed with untransduced cells at different ratios, 5 μM GCV was added, and cell proliferation was measured 4 days later. The data represent the average and standard deviation of three measurements. OD570–650, optical density at 570 to 650 nm. □, −GCV; ▵, +GCV.

DISCUSSION

We have investigated whether expression of a CKI in combination with TK can increase the efficacy of prodrug-mediated bystander cell killing. We hypothesized that growth arrest would render cells resistant to TK/GCV, allowing for more sustained prodrug conversion and diffusion to neighboring cells. This approach is well suited for diseases of cell proliferation, including restenosis, hyperplasia, and localized malignancies. We show that coordinate expression of p27 and TK increases cell killing and confers a more potent bystander effect.

Among the inhibitors of cell cycle progression that were tested, including p21, p27, p16, and Vpr, p27 showed the most effective growth arrest, despite the fact that 293 cells express the viral E1A oncoprotein (15) shown previously to inactivate p27 (20). E1A was shown to bind more strongly to the COOH- than the NH2-terminal region of p27 (20). Here, the activity of pCMVp27N, which expresses only the N-terminal part of p27, was not higher than that of pCMVp27, which expresses the full-size protein (data not shown), suggesting that expression of p27 in this highly transfectable line was sufficient to saturate limited amounts of constitutively expressed E1A. A fusion protein between p27 and TK retained both growth arrest and cytotoxic activities at levels comparable to those of the individual proteins. This fusion protein retains a nuclear localization sequence and suggests that prodrug conversion is likely equally effective whether in the nucleus or cytoplasm, where it has been localized previously (16).

As shown previously, we have found that the 65-amino-acid NH2-terminal region of p27 is necessary and sufficient for binding to cyclin-CDK complexes (19, 30). In addition, our results show that the addition of sequence downstream of p27 does not interfere with cell cycle inhibition. These findings are consistent with the nonglobular, extended structure of the NH2-terminal region of p27 proposed by Russo et al. (34). In 293 cells, pCMVp27citeTK and pCMVp27revciteTK showed comparable TK activities (Fig. 2 and 4). Cell death was not observed upon transfection of Renca cells with pCMVp27revciteTK, in contrast to pCMVp27citeTK and pCMVp27TK (Fig. 6), likely because the transfection efficiency in this cell line is lower and lysis occurs largely through the bystander effect (25). This finding is therefore consistent with the expectation that coexpression of p27 with TK would sustain TK activity and increase its potency and subsequent bystander effect.

The p27/TK fusion protein offered an alternative approach by which to combine expression of these gene products and could be used in combination with a third gene product. For example, the p15 CKI would be expected to cooperate with p27 to induce cell cycle arrest (32). Alternatively, cytosine deaminase could be used as an independent cytotoxic prodrug which could complement the activity of TK/GCV (33). Synergy has also been demonstrated between IL-2 and TK in tumor models (1, 6). The possibility of enhancing bystander cell killing at the same time that cell proliferation is inhibited suggests that this approach may also be applicable to diseases of cell proliferation, such as restenosis, or to localized malignancies such as head and neck carcinoma or sarcomas, where complete local resection is often not successful.

ACKNOWLEDGMENTS

We thank Anne Mraunac, Donna Gschwend, and Nancy Barrett for manuscript preparation.

This work was supported in part by a grant from the National Institutes of Health (HL53466). X. Danthinne was supported by a fellowship from the D. Collen Foundation. E.G.N. is an Established Investigator of the American Heart Association.

REFERENCES

  • 1.Benedetti S, Dimeco F, Pollo B, Cirenei N, Colombo B M, Bruzzone M G, Cattaneo E, Vescovi A, Didonato S, Colombo M P, Finocchiaro G. Limited efficacy of the HSV-TK/GCV system for gene therapy of malignant gliomas and perspectives for the combined transduction of the interleukin-4 gene. Hum Gene Ther. 1997;8:1345–1353. doi: 10.1089/hum.1997.8.11-1345. [DOI] [PubMed] [Google Scholar]
  • 2.Bi W L, Parysek L M, Warnick R, Stambrook P J. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther. 1993;4:725–731. doi: 10.1089/hum.1993.4.6-725. [DOI] [PubMed] [Google Scholar]
  • 3.Boehme R E. Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isoenzymes. J Biol Chem. 1984;259:12346–12349. [PubMed] [Google Scholar]
  • 4.Borrelli E, Heyman R, Hsi M, Evans R M. Targeting of an inducible toxic phenotype in animal cells. Proc Natl Acad Sci USA. 1988;85:7572–7576. doi: 10.1073/pnas.85.20.7572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Breakefield X O, DeLuca N A. Herpes simplex virus for gene delivery to neurons. New Biol. 1991;3:203–218. [PubMed] [Google Scholar]
  • 6.Chen S H, Kosai K, Xu B, Pham-Nguyen K, Contant C, Finegold M J, Woo S L. Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. Cancer Res. 1996;56:3758–3762. [PubMed] [Google Scholar]
  • 7.Colombo B M, Benedetti S, Ottolenghi S, Mora M, Pollo B, Poli G, Finocchiaro G. The “bystander effect”: association of U-87 cell death with ganciclovir-mediated apoptosis of nearby cells and lack of effect in athymic mice. Hum Gene Ther. 1995;6:763–772. doi: 10.1089/hum.1995.6.6-763. [DOI] [PubMed] [Google Scholar]
  • 8.Culver K W, Ram Z, Wallbridge S, Ishii H, Oldfield E H, Blaese R M. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science. 1992;256:1550–1552. doi: 10.1126/science.1317968. [DOI] [PubMed] [Google Scholar]
  • 9.Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau N R. Mutational analysis of cell cycle arrest, nuclear localization, and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol. 1995;69:7909–7916. doi: 10.1128/jvi.69.12.7909-7916.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ezzeddine Z D, Martuza R L, Platika D, Short M P, Malick A, Choi B, Breakefield X O. Selective killing of glioma cells in culture and in vivo by retrovirus transfer of the herpes simplex virus thymidine kinase gene. New Biol. 1991;3:608–614. [PubMed] [Google Scholar]
  • 11.Fick J, Barker F G, Dazin P, Westphale E M, Beyer E C, Israel M A. The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro. Proc Natl Acad Sci USA. 1995;92:1071–11075. doi: 10.1073/pnas.92.24.11071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Freeman S M, Abboud C N, Whartenby K A, Packman C H, Koeplin D S, Moolten F L, Abraham G N. The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res. 1993;53:5274–5283. [PubMed] [Google Scholar]
  • 13.Freeman S M, Whartenby K A, Freeman J L, Abboud C N, Marrogi A J. In situ use of suicide genes for cancer therapy. Semin Oncol. 1996;23:31–45. [PubMed] [Google Scholar]
  • 14.Gordon J W, Scangos G A, Plotkin D J, Barbosa J A, Ruddle F H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA. 1980;77:7380–7384. doi: 10.1073/pnas.77.12.7380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Graham F L, Smiley J, Russel W C, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  • 16.Haar H, Flatmark T. Evidence that deletion of coding sequences in the 5′ end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products. J Gen Virol. 1987;68:2817–2829. doi: 10.1099/0022-1317-68-11-2817. [DOI] [PubMed] [Google Scholar]
  • 17.Heyman R A, Borrelli E, Lesley J, Anderson D, Richman D D, Baird S M, Hyman R, Evans R M. Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency. Proc Natl Acad Sci USA. 1989;86:2698–2702. doi: 10.1073/pnas.86.8.2698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Luca F C, Shibuya E K, Dohrmann C E, Ruderman J V. Both cyclin AΔ60 and BΔ97 are stable and arrest cells in M-phase, but only cyclin BΔ97 turns on cyclin destruction. EMBO J. 1991;10:4311–4320. doi: 10.1002/j.1460-2075.1991.tb05009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Luo Y, Hurwitz J, Massagué J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature. 1995;375:159–161. doi: 10.1038/375159a0. [DOI] [PubMed] [Google Scholar]
  • 20.Mal A, Poon R Y C, Howe P H, Toyoshima H, Hunter T, Harter M L. Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFβ-treated cells. Nature. 1996;380:262–265. doi: 10.1038/380262a0. [DOI] [PubMed] [Google Scholar]
  • 21.Moolten F L, Wells J M, Heyman R A, Evans R M. Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene. Hum Gene Ther. 1990;1:125–134. doi: 10.1089/hum.1990.1.2-125. [DOI] [PubMed] [Google Scholar]
  • 22.Moolten F L, Wells J M. Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors. J Natl Cancer Inst. 1990;82:297–300. doi: 10.1093/jnci/82.4.297. [DOI] [PubMed] [Google Scholar]
  • 23.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  • 24.Muller D W M, Gordon D, San H, Yang Z-Y, Pompili V J, Nabel G J, Nabel E G. Catheter-mediated pulmonary vascular gene transfer and expression. Circ Res. 1994;75:1039–1049. doi: 10.1161/01.res.75.6.1039. [DOI] [PubMed] [Google Scholar]
  • 25.Ohno T, Yang Z-Y, Xu L, Jaffe M, Nabel E G, Normolle D, Nabel G J. Combination gene transfer to potentiate tumor regression. Gene Ther. 1997;4:361–366. doi: 10.1038/sj.gt.3300403. [DOI] [PubMed] [Google Scholar]
  • 26.O’Malley B W, Cope K A, Chen S H, Li D, Schwartz M R, Woo S L. Combination gene therapy for oral cancer in a murine model. Cancer Res. 1996;56:1737–1741. [PubMed] [Google Scholar]
  • 27.O’Malley B W, Jr, Sewell D A, Li D, Kosai K, Chen S H, Woo S L, Duan L. The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer. Mol Endocrinol. 1997;11:667–673. doi: 10.1210/mend.11.6.0012. [DOI] [PubMed] [Google Scholar]
  • 28.Pagano M, Tam S W, Theodoras A M, Beer-Romero P, Del Sal G, Chau V, Yew P R, Draetta G F, Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science. 1995;269:682–685. doi: 10.1126/science.7624798. [DOI] [PubMed] [Google Scholar]
  • 29.Plautz G, Nabel E G, Nabel G J. Selective elimination of recombinant genes in vivo with a suicide retroviral vector. New Biol. 1991;3:709–715. [PubMed] [Google Scholar]
  • 30.Polyak K, Lee M-H, Erdjument-Bromage H, Koff A, Roberts J M, Tempst P, Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994;78:59–66. doi: 10.1016/0092-8674(94)90572-x. [DOI] [PubMed] [Google Scholar]
  • 31.Ram Z, Walbridge S, Heiss J D, Culver K W, Blaese R M, Oldfield E H. In vivo transfer of the human interleukin-2 gene: negative tumoricidal results in experimental brain tumors. J Neurosurg. 1994;80:535–540. doi: 10.3171/jns.1994.80.3.0535. [DOI] [PubMed] [Google Scholar]
  • 32.Reynisdottir I, Polyak K, Iavarone A, Massague J. Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. Genes Dev. 1995;9:1831–1845. doi: 10.1101/gad.9.15.1831. [DOI] [PubMed] [Google Scholar]
  • 33.Rogulski K R, Kim J H, Kim S H, Freytag S O. Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene Ther. 1997;8:73–85. doi: 10.1089/hum.1997.8.1-73. [DOI] [PubMed] [Google Scholar]
  • 34.Russo A A, Jeffrey P D, Patten A K, Massagué J, Pavletich N P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-CDK2 complex. Nature. 1996;382:325–331. doi: 10.1038/382325a0. [DOI] [PubMed] [Google Scholar]
  • 35.Samejima Y, Meruelo D. ‘Bystander killing’ induces apoptosis and is inhibited by forskolin. Gene Ther. 1995;2:50–58. [PubMed] [Google Scholar]
  • 36.Schmid I, Uitteenbogaart C H, Giorgi J V. A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry. 1991;12:279–285. doi: 10.1002/cyto.990120312. [DOI] [PubMed] [Google Scholar]
  • 37.Serrano M, Hannon G J, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–707. doi: 10.1038/366704a0. [DOI] [PubMed] [Google Scholar]
  • 38.Sheaff R J, Groudine M, Gordon M, Roberts J, Clurman B E. Cyclin E-CDK2 is a regulator of p27-Kip1. Genes Dev. 1997;11:1464–1478. doi: 10.1101/gad.11.11.1464. [DOI] [PubMed] [Google Scholar]
  • 39.St. Clair M H, Lambe C U, Furman P A. Inhibition by ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Antimicrob Agents Chemother. 1987;31:844–849. doi: 10.1128/aac.31.6.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Tanner F C, Yang Z-Y, Gordon D, Nabel G J, Nabel E G. Expression of cyclin-dependent kinase inhibitors in cardiovascular disease. Circ Res. 1998;82:396–403. doi: 10.1161/01.res.82.3.396. [DOI] [PubMed] [Google Scholar]
  • 41.Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell. 1994;78:67–74. doi: 10.1016/0092-8674(94)90573-8. [DOI] [PubMed] [Google Scholar]
  • 42.Yang Z-Y, Perkins N D, Ohno T, Nabel E G, Nabel G J. The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo. Nat Med. 1995;1:1052–1056. doi: 10.1038/nm1095-1052. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES