Time-dependent changes in proliferation, DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model.

Soha A Hassan,Maike Von Harten,Charlotte Von Gall,Albrecht Piiper,Horst‐Werner Korf,Christian Schmithals

doi:10.1002/ijc.33228

Abstract

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-of-day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.

Highlights

Hepatocellular carcinoma (HCC) ranks fourth among cancer-related mortalities worldwide with a mortality rate of 8.2% (782 000 deaths)Abbreviation: CT, circadian time; DNA-DSBs, DNA-double-strand breaks; HCC, hepatocellular carcinoma; LD, light-dark; HL, healthy liver; organotypic slice cultures (OSC), organotypic slice culture; ZT, Zeitgeber time.and with high new incidence cases in 2018.1 The most common causes of HCC are related to alcohol abuse and chronic infection with hepatitis B and C viruses which are accompanied with inflamed and cirrhotic liver
The number of γ-H2AX positive cells in HCC peaked at ZT18, while in OSC their number remained high during subjective day and night
Taking this topic into consideration we have addressed three questions in the present study: (a) Do cell proliferation and DNA damage repair mechanisms show a distinct temporal pattern that would help to determine the optimal time point(s) for antimitotic therapy? (b) Does the expression of clock genes differ between normal and tumor tissue? (c) Are organotypic slice cultures an appropriate model to determine the optimal time point(s) for antimitotic therapies? The investigations were performed with a wellestablished animal model for hepatocellular carcinomas, the double transgenic c-myc/TGFα mice

Summary

| INTRODUCTION

Hepatocellular carcinoma (HCC) ranks fourth among cancer-related mortalities worldwide with a mortality rate of 8.2% (782 000 deaths). After DNA is repaired, γ-H2AX is dephosphorylated.[12,13] γ-H2AX can be used as a marker of radio-sensitivity of cancer and the normal surrounding tissues, their ability to recover from damage and the efficacy of the cellular repair process This helps to control the dosage, the effectiveness and frequency of radiation therapy in localized target.[12]. To evaluate any beneficial effect of chronotherapy, it is necessary to clarify whether cell proliferation and DNA repair mechanisms in HCC cells follow a diurnal pattern and whether this pattern differs from that in healthy liver (HL) tissue These questions are addressed in the present study in an animal model for HCC, double transgenic c-myc/TGFα mice[14] by immunohistochemical demonstration of Ki67 and γ-H2AX. The results indicate that the efficacy of antimitotic therapies depends on proper timing and that this time dependency should be evaluated further

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION