Biomedical subjects

J G DeLuca

Publications and source records attributed to J G DeLuca.

25 records · Page 2Linked to original sources

PubMed1988

A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog.

The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen with 7-EC (5- to 6-fold over control after 50 mg/kg/day for 4 days in Sprague-Dawley rats compared to approximately 2-fold for 7-EC). Since the reaction was monitored by direct fluorometry of the product, any departures from linearity under a particular set of reaction conditions (e.g. with highly induced samples) were immediately apparent. In the absence of an NADPH-regenerating system, background drift was very low (less than 0.01 fluorescent units), so the sensitivity of the assay was limited primarily by that of the fluorometer employed. This makes the assay particularly useful in situations where test material is limited, e.g. when measuring activity in cultured hepatocytes. Its simplicity, reproducibility, and response to a variety of inducing agents also make it suitable for a rapid screening assay for cytochrome P-450 induction.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1986

Malignant transformation of a preneoplastic hamster epidermal cell line by the EJ c-Ha-ras-oncogene.

We have investigated whether the activation of endogenous ras genes is associated with the immortalization or malignant transformation of primary hamster epidermal cells by chemical carcinogens. We have also asked whether transfection of a cloned c-Ha-ras oncogene (pEJ) into a nontumorigenic cell line established from hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine treatment can induce conversion to a malignant phenotype. DNA from the nontumorigenic epidermal cell line (H5-MNNG) and from two neoplastic cell lines transformed by benzo(a)pyrene was not capable of transforming NIH/3T3 cells. This result suggests that these cells do not contain an activated (mutated) ras gene. However, when H5-MNNG cells were cotransfected with pEJ and pSV2-gpt, a plasmid containing the dominant selectable marker gene Ecogpt, seven of nine clones of Ecogpt transformants formed carcinomas in nude mice and colonies in soft agar. Southern blot analysis of BamHl-digested genomic DNA from the Ecogpt-transformed clones indicated that rapid malignant transformation was associated with integration of a complete copy of the 6.6-kilobase fragment of pEJ containing the activated c-Ha-ras gene. Furthermore, DNA from the malignant clones transformed NIH/3T3 cells in a secondary transfection assay. These studies demonstrate that a mutated c-Ha-ras gene, under the transcriptional control of its normal cellular promoter, can rapidly transform a nontumorigenic epidermal cell line. This result suggests that activation of an endogenous c-ras gene can function as the final completing event in the progression of epithelial cells to the malignant phenotype. Thus, preneoplastic cell lines of both mesenchymal and epithelial origin have now been shown to be susceptible to malignant conversion by a single mutation in a c-Ha-ras proto-oncogene.

Animals↗

PubMed1984

Mutation of xeroderma pigmentosum lymphoblasts by far-ultraviolet light.

Survival and mutation were measured after UV irradiation in a human diploid B-lymphoblastoid line, XPA3, derived from a xeroderma pigmentosum patient of complementation group C. Relative to a normal human lymphoblastoid line, the XPA3 cells were more sensitive to killing as evidenced by a diminished shoulder in the survival curve and a steeper slope in the log-linear portion of the survival curve. While XPA3 cells were also more sensitive to mutation than normal cells, the data are complex. We interpret them to show a diminished threshold and a greater slope in the mutation curve at fluences which are not appreciably toxic. Examination of the 3H-TdR labeling index and rate of DNA synthesis after irradiation indicated that, at low equitoxic UV fluences (S greater than or equal to 0.85), XPA3 cells in S phase failed to slow their rate of DNA synthesis to the same extent as normal cells. Thus, fewer XPA3 than normal cells accumulated in S phase at relatively non-toxic fluences. However, at fluences which were toxic to XPA3 but not to normal cells, the early effects on DNA synthesis for both cell types were virtually indistinguishable. Our observations suggest that XPA3 cells have a reduced repair capacity for UV damage both because of a reduced rate of DNA repair and because uninterrupted passage through S phase reduced the time available for repair before fixation of both lethal and mutational damage. Furthermore, we interpret the relatively greater slopes in survival and mutation curves as an indication that, in XPA3 cells, there is a greater probability that an unrepaired lesion will result in cell death or mutation.

Cell Survival↗

PubMed1984

Construction of a plasmid containing functional Escherichia coli uvrA, B, and C genes in a configuration potentially suitable for mammalian expression.

A plasmid, pUVABC-2, was constructed that encodes functional uvrA, B, and C genes of Escherichia coli. This plasmid also contains the gpt and ampr genes for positive selection in either bacterial or mammalian systems. Each of the uvrA, B, C, and gpt genes is located between SV40 initiation and termination signals and retains the original bacterial promoters. This recombinant vector conferred a wild-type UV resistance phenotype to uvrA-, B-, and C- strains of E. coli. The results indicate that each of the uvr genes contained in pUVABC-2 function in E. coli. The plasmid is a potential biological probe for DNA repair in mammalian cells.

Animals↗

PubMed1983

Ultraviolet light-induced mutation of diploid human lymphoblasts.

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited 3H-TdR uptake into DNA. A shoulder of about 6 J/m2 and a D0 of 6 J/m2 was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m2; above 6 J/m2, complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m2 caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation. Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 J/m2), moderately toxic fluences (8.8 J/m2) and toxic fluences (17.6 J/m2). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show an enhanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.

Cell Line↗

PubMed1976

Mutation of human lymphoblasts by methylnitrosourea.

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8-12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (less than 0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 mug 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.

Cell Line↗

PubMedDate not supplied

Mutation assay in diploid human lymphoblasts: methodological aspects.

The protocol for a recently developed quantitative assay for mutation at the hgprt locus of human lymphoblasts is presented. Practical problems affecting ease of performance and reliability are discussed with the aim of making the assay available for assessment and possible use in other laboratories.

Cell Line↗