Onset of hepatocarcinogen‐specific cell proliferation and cell cycle aberration during the early stage of repeated hepatocarcinogen administration in rats

Abstract

We have previously reported that a 28-day treatment of carcinogens evoking target cell proliferation activates G1 /S checkpoint function and apoptosis, as well as induction of aberrant ubiquitin D (Ubd) expression, suggesting disruptive spindle checkpoint function, in rats. The present study aimed to determine the onset time of rat liver cells to undergo carcinogen-specific cell cycle aberration and proliferation. Animals were treated orally with a hepatocarcinogenic dose of methyleugenol or thioacetamide for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants (acetaminophen, α-naphthyl isothiocyanate or promethazine). Carcinogen-specific liver cell kinetics appeared at day 28 as evident by increases of cell proliferation, p21(Cip1+) cells, phosphorylated-Mdm2(+) cells and cleaved caspase 3(+) cells, and upregulation of DNA damage-related genes. Hepatocarcinogens also downregulated Rbl2 and upregulated Cdkn1a and Mdm2, and decreased Ubd(+) cells co-expressing phosphorylated-histone H3 (p-Histone H3) and p-Histone H3(+) cell ratio within the Ki-67(+) proliferating population. These results suggest that it takes 28 days to induce hepatocarcinogen-specific early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function as evidenced by reduction of Ubd(+) cells staying at M phase. Disruption of G1 /S checkpoint function reflected by downregulation of Rbl2 as well as upregulation of Mdm2 suggestive of sequestration of retinoblastoma protein is responsible for the facilitation of carcinogen-induced cell proliferation at day 28. Accumulation of DNA damage probably in association with facilitation of p53 degradation by activation of Mdm2 may be a prerequisite for aberrant p21(Cip1) activation, which is responsible for apoptosis.

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

“…In the present study, hepatocarcinogenic MP increased the transcript levels of Mad2l1 and Chek1 at days 28 and 90, as previously reported (Kimura et al, 2015). However, non-hepatocarcinogenic PMZ also increased the expression of these genes at day 28, while facilitating cell proliferation and cell cycle.…”

“…We have previously reported that hepatocarcinogens specifically upregulated Mad2l1, a spindle checkpoint gene (Weaver and Cleveland, 2005), and Chek1, a G 2 /M checkpoint gene that responds to DNA damage (Patil et al, 2013), after a 28-day administration, suggesting the presence of G 2 and M phase-arrested hepatocyte populations after hepatocarcinogen treatment (Kimura et al, 2015). In the present study, hepatocarcinogenic MP increased the transcript levels of Mad2l1 and Chek1 at days 28 and 90, as previously reported (Kimura et al, 2015).…”

“…In our previous short time course study of up to 28 days using rats, we have shown hepatocarcinogen-specific cellular responses suggestive of disruption in G 1 /S checkpoint and spindle checkpoint functions, as well as facilitation of cell proliferation and apoptosis at day 28 after starting administration, while no carcinogen-specific responses were observed at days 3 and 7 (Kimura et al, 2015). In the present study, the hepatocarcinogen, MP, facilitated cell proliferation and increased cell populations expressing cell cycle-related molecules and apoptosis marker at days 7 and 28 after starting administration.…”

“…We have previously reported that a 28-day administration of TAA showed a similar pattern of increases in cell proliferation, and in cell populations immunoreactive to cell cycle-related molecules and apoptosis (Kimura et al, 2015;Taniai et al, 2012b;Yafune et al, 2013b). Therefore, both TAA and MP have the potential to continuously facilitate cell proliferation and increase the number of p-Histone H3 + , TOP2A + and UBD + cells, and apoptosis by repeated administration for more than 28 days.…”

“…These results suggest that carcinogens facilitating target cell proliferation induce aberrant cell cycle control including dysfunction of M phase players, indicating the early cellular events responsible for the carcinogenesis. Furthermore, we have also recently found that such hepatocarcinogens specifically downregulated Rbl2, upregulated Mdm2 and increased the number of phosphorylated-MDM2 (p-MDM2) + cells, reflecting disruption of G 1 /S checkpoint function, as well as aberrant UBD expression at G 2 phase, after 28 days in a time-course study of 3, 7 and 28 days of administration (Kimura et al, 2015). These results suggest that it may take 28 days to direct liver cells to hepatocarcinogen-specific disruption of cell cycle regulation.…”