Immunohistochemical study of expression of p-glycoprotein and mutant p53 protein in hepatocellular carcinoma from the viewpoint of modulation of transcriptional activity of MDR1 gene

International Hepatology Communications 5 (1996) 317-325

ELSEVIER

Immunohistochemical study of expression of p-glycoprotein and mutant ~53 protein in hepatocellular carcinoma from the viewpoint of modulation of transcriptional activity of MDRl gene Mariko Department

of Internal

Medicine

Itsubo*,

1, The Jikei Minato-ku,

Gotaro

Toda

University School of Medicine, Tokyo 105, Japan

3-25-8,

Nishishinbashi,

Received 9 May 1996; revised 24 June 1996; accepted 12 July 1996

Abstract Recently, it wasreported that the promoter regionof the humanMDRl genewasa target for the ~53 tumor suppressorgeneproduct; a mutant p53 specificallystimulatedthe MDRl genepromoter. To elucidate how both proteins, p-glycoprotein and mutant p53 protein, correlatewith each other in a humancarcinomacell of HCC in vivo, the expressionof these proteinswasinvestigatedimmunohistochemically.The resultsfrom 42 casesof HCC revealed that 28 cases(66.7%) had p-glycoprotein and 12 cases(28.6%) had mutant p53 protein. Regarding the positivity rate of each protein in each histologic differentiation, that of p-glycoprotein washigher in well or moderately differentiated gradethan in poorly differentiated grade,whereasthat of mutant ~53 protein waslower in well or moderately differentiated grade.The distribution of eachprotein-positivecell wasnot alwaysuniform through the tumor sections,and the locationsof p-glycoprotein-positivecarcinomacellsand mutant ~53 protein-positivecarcinomacellswerepoorly coincidentin the serialsectionsof the samecase.

* Corresponding author. Tel: + 81 3 34331111; fax: + 81 3 34351922. 0928-4346/96/$12.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO928-4346(96)003 14-3

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It seems that ~53 protein may not directly affect the expression of p-glycoprotein, therefore p-glycoprotein overexpression in HCC could not be explained only by the direct correlation between mutational inactivation of ~53 and stimulation of transcriptional activity of MDRl gene. Keywords: p-Glycoprotein;

p53 protein; Immunohistochemistry;

Human hepatocellular car-

cinoma

1. Introduction The overexpression of p-glycoprotein [l], multidrug resistance (MDRl) gene product, is thought to be responsible for resistance to cancer chemotherapy. This membrane protein had a function of transporting various antitumor agents out of cells with a highly active efflux mechanism [2,3]. Besides, it is suggested that p-glycoprotein, normally expressed in the cell surface of epithelial cells of the adrenal gland, kidney, liver, jejunum, and colon and in capillary endothelial cells of the brain, is a transporter [4,5]. We have reported that the incidence of overexpression of p-glycoprotein is high in hepatocellular carcinoma (HCC), one of the malignant neoplasms with poor response to the chemotherapy, whether or not it was previously exposed by antitumor agents [6]. It is considered that p-glycoprotein participates in the multidrug resistant mechanism both on innate (intrinsic) and acquired resistance. The regulation by which p-glycoprotein is overexpressed in human tumors is not fully understood. It is suggested the major regulatory mechanism of p-glycoprotein overexpression in human tumors is at the transcriptional level, since MDRlRNA levels were elevated in various clinical tumors [7]. It has been shown that the p53 protein plays a role in transcriptional regulation [8,9]. Recently, it was reported that the promoter region of the human MDRl gene was a target for the p53 tumor suppressor gene product; a mutant p53 specifically stimulated the MDRl gene promoter and wild-type p53 repressed the MDRl gene promoter [lO,ll]. In the present study, to elucidate how p-glycoprotein and mutant p53 protein correlate with each other in a human carcinoma cell of HCC in vivo, their expression was investigated immunohistochemically.

2. Materials 2.1. Tumor

and methods samples

Tumor samples were obtained from 42 patients with HCC at surgical resection (16 patients) or autopsy within 5 h after their death (26 patients). Specimens were fixed in 10% formalin and embedded in paraffin. Serial sections were prepared at 4 pm from each sample for hematoxylin-eosin and immunohistochemical staining of p-glycoprotein and mutant p53 protein.

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2.2. Immunohistochemistry Immunohistochemical staining of p-glycoprotein was performed by the avidinbiotinyl-peroxidase technique using the monoclonal antibody JSB-1 (SANBIO, Amsterdam, The Netherlands) and vectastain ABC kit (Vector, Burlingame, CA). After the slides were dewaxed in xylene and rehydrated in alcohol, they were incubated in hydrogen peroxide in methanol to block endogeneous peroxidase. Each slide was incubated with normal horse serum for 20 min at room temperature and then monoclonal antibody JSB-1, the working dilution was l:lOO, was incubated on the tissue section overnight at 4°C. After incubation in biotinylated horse anti-mouse IgG (the working dilution was 1:200) for 30 min at room temperature, each slide was rinsed in phosphate-buffered saline and was incubated in the avidin-biotin horseradish peroxidase complex for 40 min at 37°C. The peroxidase was visualized with 3-3’-diamino-benzidine-tetrahydrochloride solution and then counterstained with methyl green. Immunohistochemical staining of mutant p53 staining was performed using the monoclonal antibody DO-7 (NOVOCASTRA, Newcastle, UK), the working dilution was 1:50, and the streptoavidin-ABC horseradish peroxidase procedure (IMMUNON, Pittsburgh, PA) for detection of the primary antibody. Although DO-7 is a monoclonal antibody which recognizes mutant and wild-type human p53 protein, positive nuclear immunostaining is regarded as expression of mutant p53 protein following previous reports [12,13]. Tissue-sections which were placed in a beaker filled with distilled water, were treated in a microwave oven for 10 min at 95°C for antigen retrieval in tissues [14], after blocking of endogeneous peroxidase. Each slide was incubated with protein blocking agent for 10 min to prevent background staining and incubated with monoclonal antibody DO-7 overnight at 4°C. After incubation in biotinylated universal secondary antibody reagent for 30 min at room temperature, each slide was rinsed in phosphate-buffered saline and was incubated in the streptoavidin peroxidase reagent for 30 min at room temperature. The peroxidase was visualized with 3-3’-diamino-benzidine-tetrahydrochloride solution and then counterstained with methyl green. As negative controls, an irrelevant isotype-matched monoclonal antibody or phosphate-buffered saline was substituted for each primary antibody in each procedure. Normal tissues of the adrenal gland for p-glycoprotein and a colon carcinoma known to exhibit mutant p53 protein expression using the p53 monoclonal antibody for its protein served as positive controls, and immunohistochemical staining was carried out in each with the same procedure. Cells were considered positive for immunostaining if any or all of the following staining patterns were sufficiently intense to be clearly different from the appropriate isotype controls: plasma membrane staining (p-glycoprotein) or nuclear staining (mutant p53 protein). Over 2000 cells were counted in each specimen for a positive judgment. The sample was assessed positive when more than 5% of the cells stained.

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mulysis

The results were evaluated statistically

by the chi-square test

3. Results

In the parenchymal and nonparenchymal cells of the liver tissues surrounding the tumor, p-glycoprotein was stained on the bile canalicular surface of the hepatocytes and the luminal surface of epithelial cells of the biliary ductules, although mutant ~53 protein was not stained in any cells. The results of 42 cases of HCC revealed that 28 cases (66.7%) had p-glycoprotein and 12 cases (28.6%) had mutant p53 protein. Regarding the relationship between the positivity rate and the histologic differentiation, that of p-glycoprotein was higher and that of mutant p53 protein was lower in well or moderately differentiated grade than in poorly differentiated grade (Table 1). In HCC, both p-glycoprotein-positive cells and mutant p53 protein-positive cells were not distributed uniformly and the intensity of immunoreactivity was also variable. However, the constant findings of cellular localization of each protein described below were observed; p-glycoprotein was stained on the contact surface among carcinoma cells regardless of histologic type and on the luminal surface in the pseudoglandular type in HCC, and mutant ~53 protein was stained in the carcinoma cell nuclei. In the serial sections of the same case, the location of p-glycoprotein-positive carcinoma cells and mutant ~53 proteinpositive carcinoma cells were poorly coincident (Figs. l-3).

Table 1 Incidence of p-glycoprotein

and mutant p53 protein expression in HCC n

Hepatocellular carcinoma Differentiation” Well Moderately Poorly

Positivity rate (%) p-Glycoprotein

Mutant ~53 protein

42

28 (66.7)

12 (28.6)

18 20 18

15 (83.3) 23 (79.3)b 3 (16.7)b,c

2 (11.1) 2 (6.9)d 8 (44.4)d.’

“The number of subjects was greater than the total number of patients, because tumor was composed of more than two different histologic differentiations in many cases. Values with same superscript letter are significantly correlated: ‘.“p< 0.001; dP
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Fig. 1. (A) p-Glycoprotein in HCC. Immunostaining was not observed in any cells. (B) Mutant p53 protein in the same area. Immunostaining-positive cells were densely distributed. Mutant p53 protein was stained in the carcinoma cell nucleus. Original magnification: x 100.

4. Discussion Recent studies suggested that the human MDRl gene was a target for the p53 tumor suppressor gene product from the result of CAT assay analysis; a mutant p53 protein specifically stimulated the MDRl promoter in NIH3T3 cells and wild-type p53 protein exerted specific repression in human adrenocortical carcinoma (SW13) cells [lo]. Additionally, a mechanism by which p-glycoprotein may be overexpressed in human carcinomas that also expressed mutant forms of p53 protein was also reported as a result of a study using Chinese hamster ovary (CHO) cells [ 111. The correlation with expression of p-glycoprotein and mutant p53 protein in a human HCC both in vitro and in vivo has not been known. Then, we tried to examine the expression of gene products of MDRl and mutant p53 in each carcinoma cell of HCC using the immunohistochemical technique which seemed to

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be a useful method to study the cellular localization of gene products, in order to identify how they correlate in a single cell. From our results, the incidence of p-glycoprotein was high, whereas that of mutant p53 protein was relatively low. These positivity rates were similar to the values reported in other articles [I 5- 181. Regarding the positivity rate of each protein in each histologic differentiation, that of p-glycoprotein was higher in well or moderately differentiated grade than in poorly one, whereas that of mutant p53 protein was the lower in well or moderately differentiated grade. The distribution of each protein-positive cell was not always uniform through the tumor sections, and there were few cells in which mutant p53 protein coexpressed with p-glycoprotein in the sections of mutant p53 protein positive tumor samples. Therefore, in conclusion, it seems that p53 protein may not directly affect the overexpression of p-glycoprotein in HCC.

Fig. 2. (A) p-Glycoprotein in HCC. Immunostaining-positive cells were observed through the tissue section. p-Glycoprotein was stained on the carcinoma cell surface. (B) Mutant ~53 protein in the same area. Immunostaining was not observed in any cells. Original magnification: x 100.

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Fig. 3. (A) p-Glycoprotein in HCC. Immunostaining-positive (B) Mutant ~53 protein in the same area. Immunostaining carcinoma cells (arrows). Original magnification: x 50.

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cells were partly observed (arrow k :ads). was observed in p-glycoprotein-ne

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HCC is one of the tumors originating from the tissue type which has intrinsically expressed relatively high levels of p-glycoprotein in normal tissue. The fact that the incidence of expression of p-glycoprotein in the HCC not yet exposed to antitumor agents was as high as in the HCC exposed to them [6], will suggest that p-glycoprotein participates in the multidrug resistant mechanism not only on acquired but innate resistance in HCC. Therein, the result where the positivity rate of p-glycoprotein in poorly differentiated grade was lower than any other histologic grades might be explained by there being a high tendency to lose the property of normal cells in poorly differentiated tumors compared with that in well differentiated tumors. Although our results correspond to the fact that mutational inactivation of ~53 is associated with malignant progression, p-glycoprotein overexpression in HCC could not be explained only by the direct correlation between mutational inactivation of ~53 and stimulation of transcriptional activity of MDRl gene. It is suggested that besides mutant ~53 protein, proto-oncogenes including c-Ha-Ras [lo] and c-Raf kinase [19] may modulate p-glycoprotein expression. It is considered that the regulation of overexpression of p-glycoprotein is not simple, even though ~53 acts on the MDRl expression. Further investigation would be needed to elucidate the regulatory mechanism of p-glycoprotein overexpression.

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