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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Departments of Molecular Pharmacology (M.W., C.L.M., L.C.H., M.K.D., P.M.P.) and Pharmaceutical Sciences (J.D.S.), St. Jude Children's Research Hospital, Memphis, Tennessee
Received September 6, 2002; accepted October 25, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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).
Inactivation of the p53 tumor suppressor gene, either by mutation or deletion,
has been observed in a variety of human malignancies
(Levine et al., 1991). The p53
protein can positively regulate transcription of a number of genes, including
GADD 45, p21, bax, mdm2, and cyclin G. Transcriptional
activation is mediated by protein binding to a p53 consensus motif, consisting
of two copies of a 10-bp sequence, rrrCwwGyyy, that can be separated from each
other by up to 13 bp (el-Deiry et al.,
1992). p53 can also repress expression of a number of genes,
including those involved in the response to chemotherapeutic drugs, such as
topoisomerase II, multidrug resistance (MDR1), multidrug-associated
protein (MRP1), and O6-methylguanine-DNA
methyltransferase (MGMT; Nguyen
et al., 1994; Harris et al.,
1996; Thottassery et al.,
1997; Wang and Beck,
1998). In addition, down-regulation of expression of a variety of
viral promoters has been observed, e.g., Rous sarcoma virus (RSV), SV40, and
Herpes simplex virus thymidine kinase
(Subler et al., 1992;
Zambetti and Levine, 1993).
This probably occurs as a result of p53 interacting with Sin3a
(Murphy et al., 1999).
We have exploited the ability of p53 to regulate transcription by designing
vectors that contain different gene promoters controlling expression of a
rabbit liver carboxylesterase (CE) that can convert the camptothecin analog
irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin
(CPT-11) into the potent topoisomerase I poison 7-ethyl-10-hydroxycamptothecin
(SN-38) (Potter et al.,
1998a). Expression of this CE in mammalian cells confers
sensitivity to CPT-11 both in culture and when grown as human tumor xenografts
in immune-deprived mice (Danks et al.,
1998,
1999;
Potter et al., 1998a). Hence,
selective expression of the rabbit CE in tumor cells and subsequent treatment
with CPT-11 may provide a novel method of cancer therapy. To determine whether
the expression of mutant or wtp53 could influence exogenous gene transcription
for use in viral-directed enzyme prodrug therapy (VDEPT), we assessed the
ability of different forms of p53 to regulate CE expression from both the
cytomegalovirus (CMV) or RSV promoters. Our results indicate that the
described method may prove useful in the selective treatment of human tumors
expressing mutant p53.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Briefly, they were derived by transfection of LS180 cells
with plasmids containing the CMV promoter regulating expression of either a
transdominant negative (tdn) p53, or no cDNA. They demonstrate similar growth
rates with doubling times of approximately 24 h.
The plasmids used in this study are detailed in
Table 1. Briefly, pCIneo was
obtained from Promega (Madison, WI), and plasmids containing both mutant and
wild-type p53 (pSN3 and pCMV281G) were obtained from Dr. G. Zambetti (St. Jude
Children's Research Hospital, Memphis, TN). pCMV281G differs from pSN3 by a
single point mutation at position 281 in the p53 protein.
Replication-deficient adenovirus containing a cDNA encoding a secreted form of
the rabbit liver CE under control of the RSV promoter (AdRSVrCES) was
generated by homologous recombination, and its construction and partial
characterization have been described in detail elsewhere
(Wierdl et al., 2001).
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Transient Transfections. Cells (1 x 107) were electroporated in 200 µl of phosphate-buffered saline using an electroporator (Bio-Rad, Hercules, CA) and a capacitance extender. Optimal conditions for transfection were achieved using 200 V and 960 µF for Saos-2 cells and 260 V and 960 µF for LS180 cells. All transfections contained the same total amount of plasmid DNA (25 µg) and cells were harvested after 48 h for CE activity analysis, Western analysis, or growth inhibition assays.
Carboxylesterase Assays. CE assays were performed as described
previously (Beaufay et al.,
1974; Potter et al., 1998). Briefly, cells were sonicated in
minimal volumes of 50 mM HEPES, pH 7.4, on ice and CE activity determined
using a spectrophotometric assay with o-nitrophenol acetate as a
substrate. Protein concentrations were determined using Bio-Rad protein
reagent and bovine serum albumin as a standard. Activity was expressed as
micromoles of substrate converted per minute per milligram of protein.
Growth Inhibition Assays. Growth inhibition assays were performed as
described previously (Danks et al.,
1998; Potter et al.,
1998a). Routinely, plasmid-transfected cells were harvested by
trypsinization, plated at 5 x 104 cells per well in 35-mm
six-well plates, and allowed to attach overnight. The following day, CPT-11
was diluted in fresh media and applied to cells. All drug concentrations were
repeated in triplicate. After a time equivalent to three cell doublings, the
media were aspirated and cells counted using a Multisizer II (Beckman Coulter,
Inc., Fullerton, CA). IC50 values were determined by computer
analysis of the data using the Prism program (GraphPad Software Inc., San
Diego, CA).
Adenoviral Transduction. Cells were plated at a density of 3 x
106 cells per T162 flasks and allowed to adhere overnight.
Adenovirus at a multiplicity of infection (moi) of 20 was applied to the cells
for 24 h. The following day, the media were removed and replaced, and cells
allowed to grow for up to 7 days. At 24-hour intervals, a small aliquot of
media was recovered and CE activity was monitored. For growth inhibition
studies, media were removed at day 5 and applied to untransduced LS180neo and
LS180tdn cells plated at a density of 1 x 105 cells per well
of a six-well plate. This approach was used because adenoviral transduction
can alter the growth rates of cells. The IC50 values for each cell
line with CPT-11 were then determined as described previously
(Wierdl et al., 2001).
Statistical Analysis of Data. Analysis of data were performed using Instat (GraphPad Software Inc.). For CE activities from transient transfection studies, one-way analysis of variance analyses were used to determine the statistical significance of all results, compared with each other. For the levels of secreted CE produced from adenovirus-transduced LS180 cell lines, Tukey-Kramer multiple comparison tests were performed to yield p values from data derived from each time point.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
This fragment contains DNA sequence from position –137 to +30 relative
to the initiation of transcription from the promoter (GenBank accession no.
L07624). Orientation of the transcriptional control region was confirmed by
DNA sequencing, and after large-scale preparation, a 1.7-kb EcoRI
containing the entire rCE open reading frame
(Potter et al., 1998a) was
ligated into the unique EcoRI site in the multiple cloning site. The
integrity of the resulting plasmid, pmdrRabfl, was determined by restriction
endonuclease analysis, and after large-scale preparation using endotoxin-free
reagents (QIAGEN, Valencia, CA), was used for transfection studies.
p53-Mediated Regulation of Carboxylesterase Expression Using the mdr1 Promoter. Because mutant p53 can specifically up-regulate gene transcription from mammalian promoters, including the human mdr1 gene, we assessed expression of rCE from pmdrRabfl. This plasmid was cotransfected into Saos-2 cells with vectors containing different p53 cDNAs. All experiments contained 25 µg of total DNA consisting of 20 µg of pmdrRabfl, 5 µg of mutant p53 plasmid (pCMV281G), or 1 µg of wtp53 plasmid (pSN3) + 4 µg of pCIneo. Forty-eight hours after transfection, CE activity was measured in the cell extracts. As indicated in Table 2, very high levels of CE activity were observed in Saos-2 cells after transfection with plasmid containing the CE under control of the CMV promoter. However, very little activity was seen after transfection with pmdrRabfl, even after cotransfection with mutant p53 cDNAs. We presumed that the mdr1 promoter was relatively weak in comparison with CMV and that up-regulation of CE expression sufficient to change the sensitivity of cells to CPT-11 would be unlikely.
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Regulation of Carboxylesterase Expression from the CMV Promoter. Because we were unable to use the human mdr1 promoter to regulate CE expression, we adopted a different strategy that relied upon the repression of gene transcription by wtp53. To determine whether the CMV promoter could regulate CE expression in a p53-dependent manner, we cotransfected 20 µg of pCIRabfl DNA into Saos-2 cells with plasmids containing either mutant (5 µg) or wtp53 (1 µg) cDNAs. Table 3 and Fig. 2 indicate the CE activities and a cell survival curve for a representative experiment. As indicated in Table 3, cells cotransfected with pCIRabfl and either the parent plasmid pCIneo or mutant p53 demonstrated high levels of enzyme activity. In contrast, cotransfection with plasmid expressing wtp53 resulted in marked suppression of CE expression, resulting in approximately 50% less enzyme activity. This is consistent with specific down-regulation of gene expression from the CMV promoter by wtp53. The reduction in CE activity translated into a 2.7-fold change in the IC50 value for CPT-11 with Saos-2 cells transfected with pCIneo compared with those transfected with wtp53 (Fig. 2).
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Western analysis of extracts derived from these transfections indicated
that approximately 5-fold more mutant p53 protein was present than wtp53 in
the Saos2 cells (data not shown). Because pCMV281G demonstrates no ability to
repress gene expression (Thottassery et
al., 1997; Sampath et al.,
2001), the increased amount of this protein will not influence
levels of rCE after transfection. If similar levels of wt and mutant p53 were
present, we would expect an even more marked reduction in CE activity from the
CMV promoter. These results indicate that the repression in CE activity by
wtp53 may provide a viable means to alter sensitivity of cells to CPT-11.
p53-Mediated Regulation of Carboxylesterase Expression from Adenoviral
Vectors. Because we observed regulation of CE expression from the CMV
promoter by wtp53 after transient transfection of p53 null cells, we wished to
determine whether the endogenous levels of wtp53 would be sufficient to
regulate transcription from an exogenous viral promoter. Because work by
Subler et al. (1992) indicated
that expression from the RSV promoter was downregulated 28-fold by wtp53,
nearly 20-fold more than that observed with CMV regulatory sequences, we
constructed adenoviral vectors expressing a secreted form of the rabbit liver
CE under control of the RSV promoter
(Wierdl et al., 2001).
After transduction of LS180neo and LS180tdn cells with AdRSVrCES at an moi of 50 for 24 h, the media were replaced, and CE activity in the culture media was monitored for up to 8 days. Figure 3 demonstrates that LS180tdn cells transduced with AdRSVrCES produce approximately 3 times more CE activity than LS180neo cells. These data are consistent with the hypothesis that expression from the RSV promoter is regulated by the endogenous p53 protein.
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To confirm that these levels of CE expression could confer sensitivity to CPT-11, we performed growth inhibition assays with media derived from LS180neo and LS180tdn cells, 140 h after AdRSVrCES transduction at an moi of 20. Figure 4 indicates the survival of these cell lines to CPT-11, with the IC50 values for LS180neo and LS180tdn cells being 0.97 and 0.35 µM, respectively.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Wang et al., 1994). We
reasoned that promoters that are regulated by p53 might be suitable candidates
for use in VDEPT in combination with an enzyme that can selectively activate a
drug. Based on this premise, we initially designed mammalian expression
vectors containing the rabbit liver CE under control of the human
mdr1 promoter. In reporter assays, this promoter is up-regulated when
cotransfected with plasmids expressing mutant p53
(Sampath et al., 2001).
However, the level of promoter activity was not sufficient to increase the
levels of CE expression such that sensitization to CPT-11 was observed.
Reports have indicated, however, that the degree of repression of viral
promoters by wtp53 may be sufficient to reduce gene expression to levels that
would affect drug sensitivity. Because we had already constructed plasmid
vectors containing the rCE cDNA under control of the CMV promoter (pCIRabfl;
Potter et al., 1998a), we
determined whether p53 could modulate expression of CE from this
transcriptional regulatory element. As indicated in
Table 3, a significant
reduction in CE activity occurred when these plasmids were cotransfected with
wtp53 expression vectors. To determine whether such repression was sufficient
to elicit a response after transfection with a drug-activating enzyme, we
assessed the sensitivity of cells to CPT-11 after cotransfection with pCIRabfl
and the p53 expression plasmids. We have previously demonstrated that
expression of this CE in mammalian cells confers sensitivity to the
camptothecin analog CPT-11 (Danks et al.,
1998; Potter et al.,
1998a,b).
After transfection of pCIRabfl into the p53 null cell line Saos-2 in
combination with p53 expression vectors, we observed selective down-regulation
of CE expression by the wtp53 protein (from pSN3;
Table 3). Additionally, this
abrogated the sensitivity of cells to CPT-11 conferred by the pCIRabfl vector.
This was not observed in cells cotransfected with mutant p53, indicating that
this form of the protein was abrogating the function of the endogenous wtp53.
These data indicate that a VDEPT approach using the regulated expression of
the rabbit liver CE in combination with CPT-11 may be viable therapy for human
tumors.
However, before initiating large-scale virus experiments to assess the
applicability of the CMV promoter to regulate CE expression, a search of the
literature was then performed to determine whether these were the optimum
transcriptional control sequences that could be regulated by the tumor
suppressor gene. This search identified an article presented by Subler et al.
(1992) that indicated that the
greatest level of repression of gene transcription by wtp53 was observed with
the RSV promoter. Levels of repression of gene expression using
chloramphenicol acetyltransferase reporter constructs indicated that although
the CMV promoter resulted in 8-fold less activity than control plasmids, the
RSV promoter was down-regulated 28-fold by wtp53. Because we had already
detailed the construction of adenovirus-expressing rCE under control of the
RSV promoter (AdRSVrCE and AdRSVrCES;
Wierdl et al., 2001), we opted
to use these viruses to assess p53-mediated regulation of expression in the
LS180 cell lines.
To provide a more biologically relevant system to assess the regulation of
gene expression compared with the somewhat artificial approach using
cotransfection of plasmid vectors, we opted to use adenovirus to transduce
isogenic cell lines expressing different forms of p53. AdRSVrCES, which
contains rCE under control of the RSV promoter, was used to transduce paired
cell lines that contained either the endogenous wtp53 gene or had been
transfected with a tdn p53. The tdn p53 protein forms tetramers with wtp53,
resulting in inactivation of the protein
(Wang et al., 1994), similar
to what has been observed in human tumors. We opted for this approach with
isogenic cell lines because it would reduce any variables such as the
susceptibility to cells to adenoviral transduction or the inherent differences
in the levels of transcription factors in unrelated cell types. After
adenoviral transduction, we observed less CE expression in cells containing
functionally active wtp53 (LS180neo) compared with tdn p53. Consistent with
our hypothesis, cells expressing tdn p53 did not down-regulate expression of
the CE and hence were relatively sensitive to CPT-11. Although the changes in
the IC50 values were modest (2.8-fold), it is generally considered
that relatively small changes in drug resistance and sensitivity may have
significant impact in chemotherapeutic treatment of human tumors. Hence, this
level of CE expression and reduction in the IC50 value may be
sufficient to elicit a biological response in vivo. To this end, we are
testing the potential use of these adenoviral vectors to selectively sensitize
human tumor xenografts containing different p53 mutations to CPT-11 in
immune-deprived mice.
Although adenovirus are exceptionally useful delivery vehicles for
recombinant gene expression, in studies with these vectors expressing rCE, we
have observed two mechanisms that alter the growth rates of cells, and hence
affect sensitivity or apparent sensitivity, to anticancer agents. First,
high-level expression of CE can actually stop cell growth, with cells being
arrested in the G2 phase of the cell cycle. This essentially makes
cells resistant to the toxic effects of CPT-11. Second, even transduction with
adenovirus that does not contain recombinant DNA for high-level gene
expression can significantly affect cell growth rates
(McPake et al., 1999). To
avoid these potentially complicating issues, we opted to use adenovirus
encoding the secreted rCE, and apply the media onto nontransduced cells. Any
effects caused by the adenovirus on the response of the parental cells to
CPT-11 would therefore be eliminated. This also allowed the direct measurement
of CE activity in the media from an identical population of cells during the
time course of protein expression. Hence, any variability in levels of CE
between different samples would be minimized.
Several investigators have indicated that gene therapy with virus
expressing wtp53 might be a viable approach in the treatment of human cancer
and there are clinical trials currently underway using adenovirus containing
this gene (Habib et al., 1999;
Nemunaitis et al., 2000).
However, for such approach to effective, it would be predicted that virtually
all tumor cells would need to be transduced. With the currently available
viral vector systems, this seems unlikely. The method described in this
article differs in that with an enzyme that can elicit a bystander effect with
CPT-11, i.e., the secreted rabbit CE
(Wierdl et al., 2001), only a
subpopulation of the tumor cells would need to be transduced. Neighboring
tumor cells would be killed by the SN-38 produced in the extracellular fluid
by the secreted CE. As indicated above, the effectiveness of these approaches
is being determined in animal model systems.
In conclusion, we have developed a VDEPT approach using the rabbit liver CE and CPT-11 where cytotoxicity is dependent upon the endogenous p53 status of the cell. Our results indicate that selective toxicity can be achieved in cells overexpressing mutant p53 and that a protective effect elicited by the repression of CE expression in cells expressing wtp53 can be generated. These studies demonstrate a novel approach to regulation of gene expression with a potential therapeutic application.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: bp, base pair(s); mdr, multidrug resistance; RSV, Rous sarcoma virus; SV40, simian virus 40; CE, carboxylesterase; wtp53, wild-type p53; VDEPT, viral-directed enzyme prodrug therapy; CMV, cytomegalovirus; tdn, transdominant; rCE, rabbit liver carboxylesterase; kb, kilobase(s).
Address correspondence to: Dr. Philip M. Potter, Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. E-mail: phil.potter{at}stjude.org
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  M. Wierdl, A. Wall, C. L. Morton, J. Sampath, M. K. Danks, J. D. Schuetz, and P. M. Potter Carboxylesterase-Mediated Sensitization of Human Tumor Cells to CPT-11 Cannot Override ABCG2-Mediated Drug Resistance Mol. Pharmacol., August 1, 2003; 64(2): 279 - 288. [Abstract] [Full Text] [PDF]
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-globin first
intron and the 3' acceptor site from the immunoglobulin gene heavy chain
variable region; mdr1, human mdr1 minimal promoter; neo, neomycin resistance
gene; ori, pBR322 origin of replication; SV40 Enh/Pr, SV40 enhancer and early
promoter; SV40pA, SV40 late polyadenylation site.
