Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats

Abstract

-We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and β-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G 1 /S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen-or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2 + cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D + cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens

“…Both ADAQ and TCP apparently increased the incidence of renal tumors in their 2-year carcinogenicity studies (NTP, 1993a;NTP, 1996). We have reported that leucomalachite green exerting marginal hepatocarcinogenicity did not reduce the ratio of p-Histone H3 + cells to that of Ki-67 + cells by repeated administration for up to 90 days (Kimura et al, 2015b). Therefore, disruption of spindle checkpoint function may not be induced by renal carcinogens exerting marginal carcinogenicity.…”

“…It has also been reported that the UBD-MAD2 interaction reduces the proliferating cell population at M phase, reflecting spindle checkpoint disruption (Theng et al, 2014). We have previously reported that 28-or 90-day administration of carcinogens irrespective of their potential to induce facilitation of cell proliferation causes aberrant expression of UBD at the G 2 phase and a reduction in the ratio of proliferative cells existing at the M phase, indicating disruption of spindle checkpoint function (Kimura et al, 2015a(Kimura et al, , 2015bTaniai et al, 2012b). In the present study, the renal carcinogens ADAQ and TCP facilitated cell proliferation and also reduced the ratio of UBD + cells coexpressing p-Histone H3 to the total number of UBD + cells and the ratio of p-Histone H3 + cells to that of Ki-67 + cells at day 28.…”

“…Furthermore, it is reported that human breast cancers showed high expression of MDM2 phosphorylation at Ser 166 in association with cell proliferation activity and a poor prognosis (Schmitz et al, 2006). We previously reported that hepatocarcinogens specifically increased transcript levels of Mdm2 and/or MDM2 + cells phosphorylated at Ser 166, suggestive of facilitation of degradation of p53 and subsequent disruption of G 1 /S checkpoint function by hepatocarcinogen treatment (Kimura et al, 2015a(Kimura et al, , 2015b. However, in the present study, we did not observe a carcinogen-specific increase in p-MDM2 + cells phosphorylated at Ser 166 by renal carcinogens, while mRNA upregulation of Mdm2 was found with these carcinogens, as with non-carcinogenic renal toxicant CBX from day 3.…”

“…We have previously reported that 28-day administration of hepatocarcinogens upregulated the spindle checkpoint gene Mad2l1 (Weaver and Cleveland, 2005), the G 2 /M checkpoint gene Chek1 (Patil et al, 2013), Cdkn1a encoding p21 Cip1 , and downregulated Rbl2, a gene encoding RB family protein that regulates the progression of G 1 /S phase (Cobrinik et al, 1996;Cobrinik, 2005), suggestive of an increase in G 2 and M phase-arrested hepatocyte populations and disruption of G 1 /S checkpoint function by hepatocarcinogens (Kimura et al, 2015a). However, in a time course administration study of hepatocarcinogens and hepatocarcinogenic promoters for up to 90 days, expression of Mad2l1, Chek1, Cdkn1a, and Rbl2 mRNA lacked specificity to carcinogens (Kimura et al, 2015b). In the present study, renal carcinogens also did not induce carcinogen-specific expression changes of these genes in any time points.…”

“…Furthermore, we have also recently found that some hepatocarcinogens specifically caused the downregulation of Rbl2, upregulation of Mdm2, and increase the number of phosphorylated-E3 ubiquitin-protein ligase MDM2 (p-MDM2) + cells, suggestive of disruption to G 1 /S checkpoint function, as well as spindle checkpoint dysfunction, only after 28 days of administration (Kimura et al, 2015a). Moreover, in a study of repeated administration of hepatocarcinogens for up to 90 days, hepatocarcinogen carbadox caused disruption of spindle checkpoint function after 90 days of administration, while this compound did not facilitate cell proliferation at this time point (Kimura et al, 2015b). These results suggest that carcinogen-specific disruption of spindle checkpoint function may be induced after 28 or 90 days of administration of hepatocarcinogens, which may be induced ahead of facilitation of cell proliferation at initial stage of carcinogenesis.…”