Laura Andrini

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour-bearing mice

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S‐phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S‐strain mice. After an appropriate period of synchronization, the C3H/S‐histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animals flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour‐bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour‐bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.

Circadian rhythm of VEGF expression in the liver of hepatectomized tumor bearing mice

In this study we analyzed VEGF-C expression in regenerating liver (after partial hepactectomy) of ES2 hepatocellular carcinoma bearing mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h throughout one complete circadian cycle from 26 to 50 h post-hepatectomy. Tumor samples were processed for immunohistochemistry. The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 cells (quantitative analysis). The results show that controls and tumor bearing mice have statistical differences at all analysed time points, but the maximun value of VEGF expression of treated animals is significantly higher (p < 0.01) than the control group. We can conclude that when a partial hepatectomy is made with the purpose of eradicating a hepatic tumor, the presence of possible metastasis could release factors related with cellular proliferation that could increase the possibilities of tumoral recidives.

Changes in DNA synthesis circadian rhythms in a hepatocellular carcinoma after hepatectomy

The effect of partial hepatectomy on the DNA synthesis rhythms of ES12a hepatocellular carcinoma grafted into C3H/S male mice was studied and compared with tumor growth in unoperated animal controls. The subtotal hepatectomy also changes the concentration of substances related to cellular proliferation to generate a compensatory hyperplasia in the liver. Thus, we analyzed the interaction between liver regeneration after hepatectomy and the DNA synthesis activity of the tumor. The animals were sacrificed every 4 h throughout one complete circadian cycle. All experimental groups of animals received an intraperitoneal dose of 50 mg of 5-bromodeoxiuridine per kg of body weight before being killed. Tumor samples were processed for histology and immunohistochemistry. DNA synthesis indices were expressed as labeled nuclei per 1000 nuclei. The results show that the ES12a tumor displays the DNA synthesis activity rhythm which, as in normal hepatocytes, passes through a peak during the nocturnal phase, and the surgery alters the temporal structure of the DNA synthesis indices. This fact should be considered in both neoplastic drug treatments and in growth related experiments.

VEGF EXPRESSION IN HEPATECTOMIZED TUMOR-BEARING MICE

The experiments were designed in order to study the VEGF expression in intact (group I), hepatectomized (group II), and hepatectomized-tumor bearing mice (group III) throughout one complete circadian time span. Adult male mice were used for the VEGF expression study. The statistical analysis was performed using analysis of variance (ANOVA). The results showed statistical differences in the VEGF expression between groups I and II, but the most significant differences were found between groups I and III. In conclusion, these expressions have a circadian rhythm in all groups; moreover, in group III, this expression was higher and appeared before than in the others.

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice

Tongue keratinocytes have high S‐phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S‐phase index in cell populations in tumor‐bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor‐free and tumor‐bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4‐h intervals until a circadian cycle was completed (from 26 until 50 h post‐surgery in the operated animals). Each animal was injected with 5‐bromodeoxyuridine (50 mg/kg) 1 h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean ± S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student—Newman—Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.

Circadian rhythm of VEGF expression in the liver of hepatectomized-tumor-bearing adult and young mice

Abstract We analyzed VEGF expression in regenerating liver (after partial hepatectomy) of tumor-bearing adult and young mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h, from 26 to 46 h post-hepatectomy. Liver samples were processed for immunohistochemistry. The results showed circadian variation of VEGF expression in hepatectomized mice (control group) and hepatectomized-tumor-bearing mice. The maximum value of VEGF expression was found at 16:00/30 h of day/hour post-hepatectomy (HD/HPH) in tumor-bearing young mice, and at 20:00/34 HD/HPH in the controls. In adult mice the maximum values of VEGF expression were at 16:00/30 HD/HPH in tumor-bearing mice, and at 08:00/46 HD/HPH in the controls. Young tumor-bearing mice showed significantly higher mean values than the controls. In conclusion, the presence of the tumor in the animals induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression.

Circadian rhythms of proliferation events in two mouse carcinomas

We studied the index of DNA synthesis (DNAs) of two cellular carcinomas: the hepatocellular ES12a and the mammary TN60 of mice, throughout one circadian cycle. In the results, we observed that both tumors have circadian rhythms (CRs), but the peaks of DNAs vary. Besides, the mean of DNAs along 24 h shows significative differences, the TN60 has higher values than the ES12a. These observed CR in the DNAs index in both carcinomas mean that, at least in partly, the proliferation of cancer cells can be regulated by endocrine factor as it normally occurs in ordinary cells. The big problem we can find for the chronopharmacology is that it is impossible to know in advance the rate of proliferation of each tumor.