Malcolm C. Paterson

On the quantitative relationship between O6-methylguanine residues in genomic DNA and production of sister-chromatid exchanges, mutations and lethal events in a Mer- human tumor cell line

O6-Methylguanine (m6G) is an altered base produced in DNA by SN1 methylating agents such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG). This lesion is repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT) in normal human cell lines, but is not repaired in certain human tumor lines that are termed Mex- or Mer-. Compared with repair-proficient cell lines, such repair-deficient tumor lines are hypersensitive to the production by MNNG of sister-chromatid exchanges (SCE), mutations and lethality. We report here that MNNG treatment produces 1 SCE for every 42 +/- 10 m6G formed in the genome of Mer- tumor cells, 1 6TG-resistant mutant for every 8 (range of 5-14) m6G produced statistically in the coding region of the hypoxanthine phosphoribosyltransferase gene, and 1 lethal event per 6650 +/- 1200 m6G. In addition, in vitro base mismatch incision at m6G: BrU pairs was similar to that at m6G: T pairs, the lesions that likely initiate SCE production. We conclude that m6G residues in genomic DNA are very recombinogenic as well as highly mutagenic in Mer- human tumor cells. The results are interpreted in terms of the relationship between methylation-induced SCE and G: T mismatch recognition.

Studies on the mechanism of resistance to mitomycin C and porfiromycin in a human cell strain derived from a cancer-prone individual

The mechanism of aerobic resistance to the quinone-containing anti-tumour agents mitomycin C (MMC) and porfiromycin (PM) has been investigated using non-transformed human cells. One of the cell strains used (3437T) was derived from an afflicted member of a cancer-prone family. This cell strain had been shown previously to be six times more resistant to the cytotoxic effects of these agents under aerobic but not hypoxic conditions when compared to a cell strain derived from an unrelated, normal donor (GM38). Differences could not be detected in the ability of cell sonicates prepared from either cell strain to produce alkylating species under aerobic conditions using a 4-(p-nitrobenzyl)pyridine assay. However, using 3H-labelled PM to monitor rapid drug uptake and subsequent accumulation due to drug metabolism, results were obtained indicating that the resistant cell strain (3437T) was deficient in an enzymatic pathway capable of metabolizing these compounds under aerobic but not hypoxic conditions. Dicumarol, an inhibitor of the quinone reductase DT-diaphorase (EC 1.6.99.2), decreased aerobic drug accumulation and cytotoxicity in the control cell strain, but did not alter the lack of accumulation noted in the resistant cell strain. Under hypoxic conditions, dicumarol increased cytotoxicity and drug accumulation in both cell strains. The mechanism of this enhanced cytotoxicity remains unclear. These results suggested that the resistant cells were deficient in the enzyme DT-diaphorase, a potential activator of PM. Enzymatic assays confirmed this and revealed no alterations in cytochrome P450 reductase (EC 1.6.2.4) activity or glutathione content. No protein characteristic of DT-diaphorase was detected in the resistant cell strain using a polyclonal rabbit-anti-rat antibody raised against this enzyme. Southern blot analysis using a rat DT-diaphorase cDNA probe demonstrated differences between the normal and resistant cell strains in the restriction fragment patterns. The present results are consistent with the hypothesis that decreased DT-diaphorase levels are causally associated with PM and MMC resistance in these cells under aerobic exposure conditions.

Factors Influencing Efficiency and Reproducibility of Polybrene-Assisted Gene Transfer

A systematic investigation of factors influencing the efficiency of polybrene-assisted gene transfer for both transient and stable foreign gene expression was carried out utilizing NIH 3T3 fibroblasts as prototypic recipients for the plasmid expression vectors pSV2cat and pSV2neo. While transfection cocktail composition and cell density, in addition to polybrene exposure conditions and exogenous DNA concentration, each played an important role, the key determinant to achieving excellent transfection efficiency proved to be the DMSO treatment regimen. Under optimal conditions, the yield of colonies resistant to the neomycin analog, G418, increased linearly at the rate of 10 clones/ng of input (native form I pSV2neo) DNA up to a plasmid concentration of 50 ng, whereupon the dose-response for colony recovery became semilogarithmic. The incidence of stable transformants was doubled by linearization of the vector DNA, whereas the addition of carrier DNA to the transfection cocktail was without effect until present at concentrations above 10-fold molar excess, at which point the efficacy of gene transfer declined rapidly. Combined Southern and dot-blot analyses of transformed cell DNA demonstrated that the polybrene-DMSO procedure led to the stable integration of relatively few copies of the marker gene in each transformant; the actual number varied from 1–3 to 10–15 per host genome, depending on the concentration of pSV2neo DNA added. The potential for the adaptation of this DNA transfection procedure for general use with other mammalian cell types, as well as its technical strengths and weaknesses, is discussed.

Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.

Human Cancer-Prone Disorders, Abnormal Carcinogen Response, and Defective DNA Metabolism

Insight into cancer, one of the principal scourges of modern man, has increased slowly but steadily over the years. Epidemiologists concur that most human malignancies are caused, at least in part, by environmental determinants over which an individual can exercise some control; in principle then, the disease is preventable to some extent (10). In practice, however, the goal of cancer prevention by large-scale efforts to minimize exposure to the causal agents would seem to be unattainable, as judged by societal experience with two major ‘life-style‚ factors, habitual tobacco usage and sunbathing. Although an ultimate aim is to develop other more socially acceptable prevention strategies, there exists in the interim a requirement for improved diagnostic techniques and more rational treatment protocols. Each of these approaches to cancer control will almost certainly necessitate clearer understanding of the fundamental mechanisms underlying the etiology and pathogenesis of the disease.

Preparation of Recombinant Plasmid DNA for DNA-Mediated Gene Transfer

Recombinant plasmid constructs are frequently employed in transfection experiments. With the availability of a wide spectrum of specialized and versatile eucaryotic cloning/expression vectors, investigators have been given powerful tools to expedite the elucidation of mechanisms governing gene expression, and to facilitate the identification of genes participating in diverse cellular processes (namely, metabolism, the immune response, differentiation and development, the repair of DNA damage, and malignant transformation).

Dose-dependent increase in repair of 1-β-d-arabinofuranosylcytosinedetectable DNA lesions in UV-treated xeroderma pigmentosum (group A) fibroblasts

The extent of DNA-excision repair was determined in human fibroblast strains from clinically normal and xeroderma pigmentosum complementation group A (XP-A) donors after irradiation with 254-nm ultraviolet (UV) light. Repair was monitored by the use of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of DNA synthesis, and alkaline sucrose velocity sedimentation to quantitate DNA single-strand breaks. In this approach, the number of araC-accumulated breaks in post-UV incubated cultures becomes a measure of the efficiency of a particular strain to perform long-patch excision repair. The maximal rate of removal of araC-detectable DNA lesions equalled approximately 1.8 sites/10(8) dalton/h in the normal strains (GM38, GM43), while it was more than 10-fold lower in both XP-A strains (XP4LO, XP12BE) examined. In normal fibroblasts the number of lesions removed during the first 4 h after irradiation saturated at approximately 10 J/m2. In contrast, the residual amount of repair in the excision-deficient cells increased as a linear function of UV fluence over a range 5-120 J/m2. Thus we conclude that the repair of araC-detectable UV photoproducts in XP group A fibroblasts is limited by availability of damaged regions in the genome to repair complexes.

Polybrene/DMSOAssisted Gene Transfer

Efforts to expand the current repertory of cell types amenable to transfection have often been thwarted by a common obstacle-namely, the low tolerance displayed by the recipient cells toward the gene-transfer regimen itself. As a result, several laboratories have turned to the use of synthetic polycations for delivering copious amounts of exogenous DNA to target cells without compromising their viability or clonogenicity. One of these compounds, polybrene, is mostly known for its ability to enhance the infectivity of retroviruses in culture by serving as an electrostatic bridge between the negatively charged viral particles and the anionic components residing on the surface of recipient cell membranes. Recently we, as well as others (1-4), have demonstrated that introduction of naked foreign DNA can be accomplished with high efficiency in a variety of mammalian cell types by exploiting a two-stage gene-transfer schedule in which polybrene is used first to promote the binding of DNA molecules to the target cell population, and dimethyl sulfoxide (DMSO) is then employed to permeabilize the DNA-coated cells. The method is simple to perform and has proven very effective, producing stable transfection frequencies on the order of 0.0l-0.l % with only nanogram quantities of input DNA. In a typical experiment, the process is initiated by bathing the recipient cell population in growth medium supplemented with polybrene and the transfecting DNA. Following a period of incubation during which polybrene-DNA complexes are allowed to form and attach to the cell surface, the cells are permeabilized by a brief exposure to growth medium containing DMSO in order to facilitate the uptake of the adsorbed complexes. The cells are then rinsed to remove the DMSO, and the transfected cell population is allowed to recover in fresh growth medium before being submitted to a dominant selection schedule or assayed for the transient expression of the transfected gene.

Bioreduction of 4-nitroquinoline 1-oxide in dysplastic nevus syndrome fibroblasts

Fibroblast strains 3012T and 3072T, derived from normal skin explants of two patients affected with familial dysplastic nevus syndrome (DNS), an hereditary variant of cutaneous malignant melanoma, have been reported to be abnormally sensitive to the cytotoxic and mutagenic effects of the procarcinogen 4-nitroquinoline 1-oxide (4NQO). In this communication we demonstrate that on exposure to a particular concentration of 4NQO, these same two DNS strains sustain an amount of DNA damage which is equal to (3012T) or only approximately 1.3 times greater than (3072T) that displayed by 8 control fibroblast strains established from clinically normal volunteers. Moreover, cell sonicates of 3072T display approximately 1.3-fold enhanced capacity to catalyze the reduction of 4NQO to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide, whereas sonicates of 3012T cells carry out this reaction at a normal rate. Accordingly, our results argue against the postulate that the 4NQO hypersensitivity exhibited by these DNS strains is merely due to an elevated capacity for bioreduction of the inert parent compound to a DNA-reactive derivative.