Biomedical subjects

D S Pasco

Publications and source records attributed to D S Pasco.

23 records · Page 2Linked to original sources

PubMed1990

Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene.

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.

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PubMed1989

Transient superinducibility of cytochrome P450c (CYP1A1) mRNA and transcription.

Simultaneous treatment of the rat hepatocyte-derived cell line LCS7 with cycloheximide and polycyclic aromatic compounds increased CYP1A1 (cytochrome P450c) gene transcription rate four- to sixfold and mRNA levels 20-fold relative to the levels in cells treated with inducers alone. When cycloheximide was added up to 1 hr after inducer, a similar degree of superinduction occurred. However, if cycloheximide was added at 1.5 hr or later, superinduction did not occur, even though an increased transcription rate continued at these times in cells treated with inducers alone. Thus, treatment with cycloheximide revealed two phases to the response of the CYP1A1 gene to inducer. During the early phase, inhibition of protein synthesis could amplify the effect of inducer. During the later phase, transcription rate and CYP1A1 mRNA levels remained elevated due to inducer treatment, but could not be further elevated by inhibiting protein synthesis. The superinduction of CYP1A1 mRNA was also examined in primary hepatocyte cultures and in explant cultures of three tissues. These varied substantially in their superinduction response. All of these exhibited elevated levels of CYP1A1 mRNA following simultaneous treatment with inducer and cycloheximide; transcription rate was superinduced three- to fourfold in primary hepatocytes, and CYP1A1 mRNA levels were superinduced 20- to 25-fold in both kidney and lung explants. However, the delayed addition of cycloheximide had varying effects in different culture systems. In primary hepatocytes, addition of cycloheximide as long as 4 hr after addition of inducer resulted in superinduction equal to that which occurred when these agents were added together. In contrast, adding cycloheximide only 2.5 hr after adding inducer resulted in undetectable superinduction in kidney explants and diminished superinduction 70% in lung explants. Although the time course of cycloheximide responsiveness following inducer treatment has been studied in detail only in LCS7 cells, it appears that the length of the cycloheximide-responsive phase is different in different cell culture systems in in explants from different tissues. This may be related to the previously reported tissue-specific differences in CYP1A1 gene expression.

Animals↗

PubMed1989

Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes.

An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times. This procedure was successful with reporter genes linked to three different eukaryotic promoters. These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene. This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.

Animals↗

PubMed1988

Transcriptional and post-transcriptional regulation of the genes encoding cytochromes P-450c and P-450d in vivo and in primary hepatocyte cultures.

In both primary cell cultures of rat hepatocytes and in liver, polycyclic aromatic hydrocarbons (PAHs) were found to influence the accumulation of the cytochrome P-450c and P-450d mRNAs by both transcriptional and post-transcriptional mechanisms. Following treatment with PAHs, cytochrome P-450c mRNA levels increased approximately 100-fold in both hepatocyte cultures and in liver, while transcription rates, measured by run-on transcription of isolated nuclei, increased 3-fold in hepatocyte cultures and 10-fold in liver. The difference in the -fold increases of mRNA level and transcription rate suggests that post-transcriptional, as well as transcriptional, mechanisms contributed to the regulation of cytochrome P-450c mRNA levels. Following treatment with PAHs, cytochrome P-450d mRNA levels increased 200-fold in hepatocyte cultures and 70-fold in liver, while transcription rates remained unchanged in hepatocyte cultures and increased only 1.7-fold in liver. This suggests that post-transcriptional mechanisms were of primary importance in regulating cytochrome P-450d mRNA levels. The newly developed hepatocyte primary cell culture system used in these studies differs from previously reported systems in that the cytochrome P-450d gene, as well as the cytochrome P-450c gene, were expressed in response to PAHs. In this cell culture system the regulation of these two genes was quite similar, although not identical, to that found in liver. The mechanisms controlling the tissue-specific expression of the genes encoding cytochromes P-450c and P-450d were also examined. The cytochrome P-450c mRNA was found in kidney, heart, and lung, as well as in liver, of PAH-treated rats, while the mature cytochrome P-450d mRNA was detected only in liver. The substantial increase in cytochrome P-450c mRNA in kidney in response to beta-napthoflavone was not associated with a detectable change in the transcription rate of cytochrome P-450c gene, indicating that cytochrome P-450c mRNA levels must be regulated primarily post-transcriptionally in kidney. Even though mature cytochrome P-450d mRNA could not be detected in kidney, the cytochrome P-450d gene was transcribed at a substantial rate in this tissue; therefore, the lack of accumulation of mature cytochrome P-450d mRNA in kidney must have been due to post-transcriptional control.

Animals↗

PubMed1985

Synthesis and degradation of 3-methylcholanthrene-inducible cytochromes P-450 and their mRNAs in primary monolayer cultures of adult rat hepatocytes.

We used primary nonproliferating cultures of adult rat hepatocytes to investigate the regulation of P-450c and P-450d, immunochemically related protein products of separate cytochromes P-450 genes that are coinduced by 3-methylcholanthrene and related compounds. In cultures of hepatocytes prepared from untreated rats and incubated in media containing 3-methylcholanthrene, beta-naphthoflavone, 3,4,3',4'-tetrachlorobiphenyl, and Aroclor 1254 (a mixture of chlorinated biphenyls) there was a 5- to 15-fold accumulation of P-450c protein (quantitated by immunoblotting), accompanied by an increased rate of P-450c synthesis (measured as incorporation of [3H]leucine into immunoprecipitable protein) and an increased amount of P-450c mRNA hybridizable to a specific cloned cDNA (p210). In contrast, there were no increases in the concentration of P-450d protein, its rate of synthesis, or the amount of P-450d mRNA hybridizable to its specific cDNA (p72). Similarly, when "preinduced" hepatocytes (isolated from rats treated with Aroclor 1254) were incubated for 4 days in culture medium, the amount of P-450c, its rate of synthesis, and the amount of P-450c mRNA remained elevated, whereas synthesis of P-450d and the amount of P-450d mRNA fell precipitously to less than 10% of the initial values despite the presence or absence of Aroclor 1254 or of isosafrole in the medium. However, the loss of P-450d protein in these cultures was almost completely prevented when isosafrole was added to the culture medium and was partially prevented when safrole, Aroclor 1254, and 3,4,5,2',4',5'-hexachlorobiphenyl, but not 3-methylcholanthrene, beta-naphthoflavone, or 3,4,3'4'-tetrachlorobiphenyl, were in the culture medium. Moreover, in similar cultures of "preinduced" hepatocytes that were pulse-labeled with [3H]leucine, the presence of isosafrole in the culture medium extended the apparent half-life for loss of radioactivity in immunoprecipitable P-450d to a value of 72 h (3-fold longer than in standard medium) but was without effect on the rate of disappearance of radiolabeled P-450c. We conclude that control of P-450d degradation is an important factor in the regulation of this hemoprotein and that induction of P-450c and P-450d proceed by separate pathways that are spontaneously divorced under standard conditions for primary culture of adult rat hepatocytes.

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