DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): host cell reactivation of damaged plasmid DNA

cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, we have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSVcat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The parent cell line L1210/0 resembled repair-deficient cells in that about one adduct per cat gene eliminated expression. In three resistant L1210 cell lines, 3-6-fold higher levels of damage were required to produce an equivalent inhibition. This did not correlate with the degree of resistance as these cells varied from 10- to 100-fold resistant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of an enhancerless gene by chromosomal integration.

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.     

Effect of DNA damage on the expression of the chloramphenicol acetyltransferase gene after transfection into diploid human fibroblasts.

The activity of the chloramphenicol acetyltransferase (cat) gene after transfection into human fibroblasts has been measured following treatment of the plasmid pRSVcat with either restriction enzymes or ultraviolet light. Restriction enzymes producing single cuts in the plasmid inactivated the expression of the cat gene whether the enzymes cut the plasmid inside the coding region of the gene or several kilobases away from the gene. Ultraviolet light produced a dose-dependent inactivation of the gene. The inactivation curve was steeper if the recipient cell strain was derived from a patient with xeroderma pigmentosum. The findings with this transient expression system contrast with previously reported results of experiments using plasmids which transform cells stably by integrating into the cellular genomic DNA.     

Regulation of the inducible chloramphenicol acetyltransferase gene of the Staphylococcus aureus plasmid pUB112.

Analyses of deletion mutants of the gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112 revealed a regulatory region, which is indispensable for Cm-inducible cat gene expression, located 70 bp in front of the CAT-coding sequence. This region consists of a possible ribosome binding site followed by an open reading frame coding for a peptide of nine amino acids and overlaps partially with an inverted repeat capable of forming a stem-loop structure. Deletion of the ribosome binding site and of parts of the open reading frame abolishes inducibility and results in a low-level cat gene expression, if the inverted repeat remains intact. Deletion of the 5' part of the possible stem leads to high-level constitutive CAT synthesis. The inverted repeat, therefore, exhibits negative control on cat gene expression whereas the preceding ribosome binding site is needed to enhance CAT synthesis in the presence of an inducer. These results suggest that translation of a leader peptide is a prerequisite for Cm-induced cat gene expression and that ribosome stalling on cat leader mRNA caused by Cm opens the stem-loop structure thereby releasing its negative effect on CAT synthesis.     

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.     

Expression of prokaryotic genes after transfection of X-irradiated plasmids into primate cells

Expression of the prokaryotic gene for chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT) in primate cells transfected with X-irradiated plasmid pSV2CAT was determined in transient expression assays. CAT expression did not depend upon the presence of supercoiled plasmids, but relaxed circular forms were essential. X-ray conversion of relaxed circles to linear forms paralleled the loss of CAT expression, with identical D0's in the first part of dose-response curves. X-ray-induced loss of supercoiled forms was complete at much lower doses. The D0 for inactivation of CAT expression by X irradiation of the plasmids in 1 mM Tris buffer was 270 Gy; it was 13 Gy for plasmids irradiated in water. The D0's for conversion of pSV2CAT to relaxed circle forms were only one-seventh as large as the D0's for CAT inactivation after X-ray in water or in 1 mM Tris buffer. Expression of the CAT gene in some representative repair-deficient human fibroblasts transfected with X-irradiated pSV2CAT was less than in monkey CV-1 cells or cell lines from normal human subjects. These results demonstrate a novel means to study low levels of X-ray damage in DNA correlating specific X-ray damage in the DNA with expression of the gene in unirradiated primate cells.     

Transcriptional trans-activating function of hepatitis B virus

The ability of hepatitis B virus (HBV) to stimulate the expression of a cellular gene was investigated by using a transient-expression system. A plasmid in which the expression of the bacterial chloramphenicol acetyltransferase (cat) gene had been placed under the control of the DNA sequences that regulate the expression of the human beta-interferon gene was constructed. In Vero cells, cotransfection of the 2.7-kilobase BglII DNA fragment of HBV together with the test plasmid containing the cat gene resulted in stimulation of the expression of the cat gene. This HBV DNA fragment was specific in its trans-activation; no significant stimulation of CAT activity was observed in constructs when the promoter and enhancer elements were derived from the murine sarcoma viral long terminal repeat, Rous sarcoma virus, BK virus, or simian virus 40. Results of subcloning of the HBV DNA fragment indicate that the trans-activating function resides in a 944-base-pair EcoRV-BglII DNA fragment of the HBV genome that contains the X structural gene and its promoter element. Removal of the promoter from the X structural gene resulted in loss of the trans-activating function. A frameshift mutation within the X gene region also eliminated the trans-activating activity. These results suggest that the X antigen could play a role in HBV infections by activating the expression of cellular genes.     

Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein.

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B lymphocytes, however, the vector was not replicated, nor did it express the CAT gene.     

Human P45IA1 upstream regulatory sequences expressing the chloramphenicol acetyltransferase gene. Effect of Ha-MSV enhancer and comparison of transient with stable transformation assays

Upstream sequences of the human P450IA1 gene were inserted into a promoterless expression vector (pSVO-cat) containing the chloramphenicol acetyltransferase (CAT) gene, with and without the Harvey murine sarcoma virus (Ha-MSV) core enhancer, and either plasmid was transfected into human breast carcinoma MCF-7 and MDA-231 and mouse hepatoma Hepa-1 cell lines. In most instances constitutive and inducible CAT activities in the transient CAT expression assay were similar (within 3-fold) to those in the stable transformation CAT assay (selection of G418-resistant colonies following co-transfection with pSV2-neo). In the case of Ha-MSV-containing constructs stably integrated in the two human breast cancer lines, however, CAT expression was more than two orders of magnitude greater than that transiently expressed in these cells. Since the major difference between these two assays is plasmid copy number, these data suggest the presence of limiting amounts of tissue-specific positive-control enhancer-binding factor(s) in the breast carcinoma cell lines.     

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Summary Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150g ml^–1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm^–1 (960 F). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.NRCC No. 30491     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli

A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.     

The role of DNA repair in resistance of L1210 cells to isomeric 1,2-diaminocyclohexaneplatinum complexes and ultraviolet irradiation

Resistance to cisplatin in several murine leukemia L1210 cell lines is due to enhanced DNA repair. Other platinum complexes, particularly those containing 1,2-diaminocyclohexane (DACH) are of interest as they effectively kill both sensitive (L1210/0) and cisplatin-resistant (L1210/DDP) cell lines. An L1210/DACH cell line has been developed that is preferentially resistant to DACH-Pt complexes. In the current experiments, we investigated the role that DNA repair has in resistance to DACH-Pt compounds. The DACH ligand exists in 3 isomeric forms which exhibit markedly different activities in the various resistant cell lines. Generally, R,R-DACH-Pt was the most effective isomer. DNA repair was assayed by host-cell reactivation of platinated pRSVcat. DNA damage induced by all the isomeric DACH-Pt-SO4 complexes markedly reduced CAT expression in sensitive L1210/0 cells. One adduct per transcribed strand of the cat gene inhibited CAT expression demonstrating that the sensitive cells exhibited no detectable DNA repair. All the resistant cell lines reactivated the plasmid DNA whether damaged with cisplatin or any of the 3 DACH-Pt isomers. Therefore, resistance to both cisplatin and DACH-Pt appears to be mediated by enhanced DNA repair, but the level of reactivation of the transfected plasmid did not correlate with the toxicity of each analogue. These results suggest that some additional event(s) is responsible for the substrate specificity of repair of genomic DNA. These resistant cell lines also exhibited resistance to UV irradiation but this was much less than, and did not correlate with the degree of resistance to either cisplatin or DACH-Pt. However, there was a good correlation between resistance to UV irradiation and reactivation of UV-damaged plasmid DNA. This enhanced reactivation suggests that enhanced repair may be the sole reason for the resistance to UV irradiation.     

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat.

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.