Ribonucleotide reductase: a possible target for orotic acid induced mitoinhibition in normal hepatocytes in primary culture

Manjeshwar, Sharmila; Pichiri, Giuseppina; Coni, P.; Rao, Prema M.; Rajalakshmi, S.; Sarma, D.S.R.

doi:10.1016/0304-3835(93)90257-a

Cited by 5 publications

(6 citation statements)

References 14 publications

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“…The degradation of RNR transcripts may not reflect general degradation of mRNAs, because OA treatment did not appear to influence the stability of mRNA transcripts of GAPDH (a housekeeping gene) or Gal-Tase (an induced gene). Interestingly, while the expression of some cell cycle-related genes like c-myc, c-fos, and Ha-ras is not significantly affected by OA in vitro [32] or in vivo (this study, data not shown), the stability of the transcripts for DHFR and TS appeared to be decreased by OA treatment (Figure 4). Therefore, the question that arises from these studies is how nucleotide pools affect the stability or degradation of RNR mRNA transcripts.…”

Section: Discussioncontrasting

confidence: 61%

“…The values are the means ± SD from four rats, each assayed in triplicate. lier results from hepatocytes in primary culture [32], the in vivo expression of c-myc and Ha-ras was not inhibited by OA treatment (data not shown). These results suggested that the inhibitory effect of OA may not be a general one on cell cycle-related genes but may be more closely related to the expression of those genes whose products are required for DNA synthesis.…”

Section: Resultsmentioning

confidence: 53%

“…The blots were baked in a vacuum oven for 2 h at 80°C and prehybridized at 42°C overnight as described previously [32]. For hybridization, the radioactive RNA samples obtained above were denatured by boiling for 10 min and quenched on ice.…”

Section: Binding Of Target Plasmids To Nitrocellulose and Hybridizationmentioning

confidence: 99%

“…Electrophoresis of RNA in formaldehyde-agarose gels, transfer to nitrocellulose filters, and northern hybridization were performed as described earlier [32]. The probes for the M1 and M2 subunits of RNR were kindly supplied by Dr. L. Thelander (University of Umea, Sweden); probes for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ATCC 57090) and mouse dihydrofolate reductase (DHFR, ATCC 37295) were from the American Tissue Type Collection (Manassas, VA).…”

Section: Electrophoresis Of Rna and Northern Hybridizationmentioning

confidence: 99%

“…One sample per blot was then injected into a bag containing a blot, such that each bag had the same amount of radioactivity (usually 5 × 10 6 cpm/mL). Hybridization was performed at 42°C for 72 h. The blots were then washed and subjected to autoradiography at -70°C as described earlier [32].…”

Section: Binding Of Target Plasmids To Nitrocellulose and Hybridizationmentioning

confidence: 99%

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The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

et al. 1999

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Our earlier studies have shown that in normal hepatocytes, orotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and after two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA induces an imbalance in nucleotide pools (specifically, an increase in uridine nucleotides, including deoxyuridine nucleotides, and a decrease in adenosine nucleotides, including ATP) and creation of this imbalance is crucial for the mitoinhibitory effects of OA, we hypothesized that ribonucleoside diphosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the inhibition of DNA synthesis by OA. To test this hypothesis, we subjected male Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the presence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneally at the time of two-thirds PH). The rats were killed at different times later, and their livers were processed for analysis of levels of RNR enzyme activity, protein, and mRNA transcripts. The results obtained indicated that treatment with OA resulted in a near-100% inhibition of RNR induced by two-thirds PH in rat liver, as monitored by enzyme activity and protein level. Furthermore, this inhibition was paralleled by a decrease in the mRNA transcripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indicated that this decrease in the levels of mRNA transcripts could not be attributed to an effect on transcription. However, administration of OA 20 h after two-thirds PH, when RNR mRNA transcripts were maximally induced, resulted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by two-thirds PH, at the levels of enzyme activity, protein, and mRNA transcripts, and the decreased levels of mRNA transcripts appeared to be due to increased degradation of the transcripts.

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Section: Discussioncontrasting

confidence: 61%

Section: Resultsmentioning

confidence: 53%

Section: Binding Of Target Plasmids To Nitrocellulose and Hybridizationmentioning

confidence: 99%

Section: Electrophoresis Of Rna and Northern Hybridizationmentioning

confidence: 99%

Section: Binding Of Target Plasmids To Nitrocellulose and Hybridizationmentioning

confidence: 99%

See 3 more Smart Citations

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

et al. 1999

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Inhibition ofin vivorat liver regeneration by 2-acetylaminofluorene affects the regulation of cell cycle-related proteins

1998

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The effects of dietary 2-acetylaminofluorene (2-AAF) on cell cycle-related proteins was studied in regenerating livers from male Wistar rats. The levels of cyclins, cyclin dependent kinases (cdks), and related proteins were studied at different times during the first cell cycle after partial hepatectomy (PH). The frequency of proliferation cell nuclear antigen (PCNA)-positive nuclei, a marker of S phase progression, was almost zero during the first 27 hours after PH in the mitoinhibited 2-AAF-treated rats, while about 50% of the nuclei were labeled 24 hours after PH in control animals. Accordingly, Western blot tests showed markedly elevated PCNA protein levels from 18 hours to the end of S phase in untreated animals but no upregulation in response to 2-AAF. Compared with control animals, animals treated with 2-AAF showed increased levels of cdk 4 and cyclin D 3 from 12 and 15 hours after PH, respectively, and altered cyclicity in cyclin D 3 expression. No effects on cyclin E were observed, while the increase in cdk 2 levels in control animals during late G 1 /S (15-27 hours) was abolished by 2-AAF. p53 was induced by 2-AAF treatment during the same period, with a peak at 24 hours. The protein detected with p21 antibodies was highly expressed in unstimulated hepatocytes in control animals, and further increased by 2-AAF. The expression was sustained until 15 hours after PH in control rats while 2-AAF-treated animals lacked detectable protein during this period; however, a transient increase was observed at 21 hours. Thus, 2-AAF affects several parameters of cell cycle regulation of possible relevance for its inhibitory effects on hepatocyte proliferation in vivo. (HEPATOLOGY 1998;27:691-696.)Many liver tumor promoters such as 2-acetylaminofluorene (2-AAF), phenobarbital, orotic acid, clofibrate and ethinylestradiol have inhibitory effects on proliferation of hepatocytes in vivo during long-term treatment, 1-5 which is believed to be an important aspect of the promotion mechanism. In the classical resistant hepatocyte model (RH-model) for rat liver carcinogenesis, where promotion is performed by dietary 2-AAF treatment combined with a partial hepatectomy (PH), the selective growth advantage of preneoplastic foci during treatment has been suggested to be caused by the inhibited proliferation of hepatocytes surrounding the lesions, combined with focal resistance to 2-AAF-induced inhibition. 1 This makes the putatively preneoplastic foci, in contrast to the surrounding tissue, able to respond to the proliferative stimulus provided by the PH. The mitoinhibitory effects of 2-AAF are caused by the formation of sulfated metabolites of N-hydroxyacetylaminofluorene, 6 which is low in the lesions compared with the surrounding hepatocytes. 7 These sulfated metabolites give rise to two different Nacetylated adducts causing major distortion of the DNA structure, and lead to an efficient DNA replication block both in vitro and in vivo. 6,[8][9] The molecular mediators of the inhibitory signals generated by 2-AAF are not known....

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Sequential histopathological analysis of hepatocarcinogenesis in rats during promotion with orotic acid

et al. 1994

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Ribonucleotide reductase: a possible target for orotic acid induced mitoinhibition in normal hepatocytes in primary culture

Cited by 5 publications

References 14 publications

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

Inhibition ofin vivorat liver regeneration by 2-acetylaminofluorene affects the regulation of cell cycle-related proteins

Sequential histopathological analysis of hepatocarcinogenesis in rats during promotion with orotic acid

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