Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor...

Vi rchows Archiv B Cell Pathol (1991 ) 60 : 133-138 Virchows Archiv B Cell Pathology Including Molecular Pathology

�9 Springer-Verlag 1991

Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor lesions Valeska Vollrath 1, Jos~ Chianale 1, Sergio Gonzalez 2, Ignaeio Duarte 2, Leonardo Andrade 2, and Luis lbafiez 1

i Departamentos de Gastroenterologia y Anatomia Patol6gica, ~ Facultad de Medicina, Pontificia Universidad Cat61ica de Chile

Received August 15 / Accepted November 29, 1990

Summary. Overexpression of the Multiple Drug Resis- tance gene (MDR1) has been proposed as a major mechanism related to both intrinsic and acquired resistance to chemotherapeutic agents. The gene product is a membrane protein (P-glycoprotein), that acts as an energy- dependent drug efflux pump decreasing drug accumulation in resistant tumor cells. We have characterized MDR1 and P-Glycoprotein expression in human gastric adenocarcinoma and in precursor lesions. MDR1 mRNAs, analyzed by dot-blot technique, were detected in 9 of 10 non-tumoral gastric mucosae and in 8 of 10 gastric adenocarcinomas. Immunohistochemical analysis, using the MRKI6 monoclonal antibody, revealed heterogeneous expression of P-Glycoprotein in individual cells. The P-Glycoprotein was found on the surface of cells of gastric areas with intestinal metaplasia subtype III. This type of intestinal metaplasia, also called "colonic metaplasia", has been strongly associated with a high risk for the development of gastric cancer. The fact that the P-Glycoprotein was detected in this precursor lesion is consistent with the intestinal metaplasia- dysplasia and carcinoma sequence proposed in the histogenesis of this tumor. The finding that P-Glycoprotein was heterogeneously expressed in malignant cells of some gastric adenocarcinomas also suggests that this transporter system probably contributes to primary and secondary multidrug resistance in this neoplasm.

Key words: Multidrug resistance gene - P-glycoprotein - Gastric cancer

Introduction

The molecular mechanisms of multiple drug resistance have been studied extensively in mammalian cell culture.

Offprint requests to : J, Chianale, Departamento de Gastroenterolo- gia, Facultad Medicina, Pontifieia Universidad Catolica, Casilla 114-D, Santiago, Chile

Tumor cell lines, selected for resistance to a large group o.f lipophylic cytotoxic compounds, show an increased expression of the Multiple Drug Resistance (MDR1) gene, which encodes a 170000 dalton plasma membrane glycoprotein (P-glycoprotein) (Kartner et al. 1983). This membrane protein is believed to be responsible for multiple drug resistance (Gros et al. 1986; Fojo et al. 1987; Goldstein et al. 1989; Gottesman et al. 1989), function- ing as an energy-dependent drug efflux pump that de- creases drug accumulation in resistant cells (Willingham et al. 1986; Hamada et al. 1986). These multidrug-resistant cell lines usually contain an amplification of the MDRI gene, which is transcribed into a 4.5-kilobase mRNA (Roninson et al. 1986).

Recent studies have revealed high levels of MDR1 expression in intrinsically drug-resistance tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas. The consistent association of MDR1 gene expression with several intrinsically resistant cancers and the increased expression of this gene in tumors with acquired or secondary drug resistance indicate that the MDR| gene contributes to multidrug resistance in many human tumors (Goldstein et al. 1989).

Gastric adenocarcinoma is a public health problem in high-risk cancer areas like Japan and Chile (Correa 1986) because of its high incidence and poor prognosis. The contribution of chemotherapy to the surgical treat- ment of gastric carcinoma is unsufficient, because most of the patients exhibit only a partial response following administration of combination chemotherapy (Mac- Donald et al. 1982).

The precancerous stages of gastric adenocarcinoma constitute a very complex and still unclear process. Mor- phological and histochemical studies suggest a possible intestinal metaplasia-dysplasia-carcinoma sequence in the histogenesis of gastric adenocarcinoma, particularly in the intestinal type of gastric cancer (Correa 1983; Filipe and Jass 1986). While dysplasia is a well recog- nized morphological marker of premalignant change in the gastric mucosa, the role of intestinal metaplasia in

the his togenesis o f gast r ic c a r c i n o m a is still a m a t t e r o f cont roversy . O u r p rev ious obse rva t ions p e r f o r m e d in au topsy mate r i a l f rom a Chi lean p o p u l a t i o n (Dua r t e et al. 1984) indica te tha t in tes t ina l me tap l a s i a cons t i tu tes a very c o m m o n f inding in the s tomach (occur r ing in more than 75% o f the cases s tudied) and this d a t a is in ag reemen t wi th o the r repor t s f rom high-r i sk cancer areas ( K u b o et al. 1971). F o r these reasons , the in tes t ina l me tap l a s i a s u r r o u n d i n g mos t gast r ic a d e n o c a r c i n o m a s and the in tes t inal m e t a p l a s i a obse rved in a t roph ic gastr i - tis have been p r o p o s e d as the mos t sensit ive ind ica to r o f gast r ic cancer ( M o r s o n et al. 1980).

In the cur ren t s tudy, we examined M D R 1 gene express ion and P -g lycopro te in d i s t r i bu t ion in h u m a n gastric c a r c i n o m a in o rde r to p rov ide i n f o r m a t i o n re la ted to the mechan i sms o f c h e m o t h e r a p y resis tance in this tumor . We also charac te r i zed the express ion o f M D R 1 and P -g lycopro te in in p recance rous lesions in an a t t e m p t to p rov ide add i t i ona l evidence to the in tes t ina l m e t a p l a - s i a - ca rc inoma sequence p r o p o s e d in the his togenesis o f this cancer .

Material and methods

Specimens. Samples were examined from 22 primary gastric carcinomas surgically excised at the Clinical Hospital of the Catholic University Medical School, from patients who had not received any anti-cancer drug. Tumoral and non-tumoral gastric mucosa samples were rapidly frozen under liquid nitrogen after removal and kept at - 7 0 ~ until processed. Non-tumoral samples were taken at least 5 cm from the border of the neoplasm.

RNA extraction. Total cellular RNA was extracted as described by Chomczynski and Sacchi (1987). The purity of the RNA was analyzed spectrophotometrically and its integrity was checked on non-denaturated 1% agarose minigel. Only ten samples in which the ribosomal RNAs appeared undegraded were analyzed.

MDR1 hybridization probes. The probe 5A of the MDR1 cDNA, which includes coding regions of the middle third of a full-length cDNA (Fojo et al. 1987), was cloned into pGEM4 (pHDR5A) and used for all the hybridizations. The probe was labeled to high specific activity (2 • 10 9 dpm/p.g DNA) with [~_s2p] dCTP (Amer- sham Corp.; 3000 Ci per raM) using the "oligolabeling'" method (Klenow fragment, Amersham Corp.) (Feinberg and Vogelstein 1984). In some experiments, the hybridizations were also performed using a [7-32P] CTP-labeled RNA probe, obtained after linearizing the pHDR5A with Pvu II and using SP6 RNA polymerase (Melton et al. 1984).

Qualitative assessment of the MDR1 mRNA : northern blot analysis. The total cellular RNA was electrophoresed in denaturing 1% agarose-6% formaldehyde gels (Shen et al. 1986). RNA (10 ~tg) were loaded per lane. After electrophoresis, the separated RNAs were transferred to nitrocellulose filters (BA 85, Schleicher and Schuetl, Keene, Nil) and fixed by baking the filters under vacuum at 80 ~ C for 2 h. The filters were pre-hybridized in 0.1 ml/cm / of a solution containing 50% formamide, 10 • Denhardt, (1 • : 0.02% ficoll, 0.02% polyvinylpirrolidone, 0.02% bovine serum albumine), 0.1% SDS, 5 x SSC, (1 x : 0.15 M sodium chloride, 0.15 M sodium citrate, pH 7), and 100 pg/ml of salmon sperm DNA. The pre- hybridization was carried out at 42 ~ C for 2 h. The hybridization was performed overnight at 42 ~ C in a similar solution containing also 10% dextran sulfate and the 32p-labeled probe. After hybridization, the filters were washed three times with 2xSSC, 0.1% SDS and once with 0.1 x SSC, 0.1% SDS at 50 ~ C for 20 min each

wash. Analysis of autoradiographs were performed using Kodak X-Omat AR film between two intensifying screens at - 7 0 ~ C.

Quantitative assessment of MDR1 mRNA : dot blot analysis. Mea- surements of the relative content of specific mRNAs were performed by dot-blot hybridization. Total RNA was denaturated in 50% formamide, 2.2% formaldehyde, 1 • (40raM MOPS, 10 mM sodium acetate, 1 mM EDTA, pH 7.5) at 65 ~ C for 5 min and cooled on ice. The RNA solution was adjusted with 20 • SSC to a final concentration of 4 • SSC and spotted on nitrocellulose filters using a Minifold I system (Schleicher and Schuell). Each sample studied was spotted in duplicate. The baked filters were prehybridized, hybridized and washed in similar conditions as described previously for Northern blotting. RNA extracted from kidney and hypernephroma tissues, which contains relatively high levels of MDR1 transcripts (Fojo et al. 1987a), were also applied to the same blot as a positive control. Immediately after the autoradiographs were obtained, spots on filters were cut out and counted in a liquid scintillation counter (LKB, 1209 Rackbeta) and the background corresponding to non-specific hybridization sub- stracted. The relative content of MDR1 mRNAs was expressed as pg of cDNA bound per 10 [.tg of total RNA spotted (Papavasi- liou et al. 1986).

Immunohistochemical analysis. The immunohistochemical analysis of the MDR1 gene product was performed using the streptavidin- biotin complex staining procedure in frozen sections. Cryostat sections 4 to 6 ~tm-thick were obtained from 12 tumoral and non- tumoral samples and dried overnight. The sections were fixed in acetone for 10 rain prior to staining. To block endogenous peroxidase activity, incubation was performed in 1% aqueous solution of H202. The non-specific reaction was blocked by incubating the sections with normal rabbit serum 1 : 20 in TBS buffer (50 mM Yris-C1, pH 7.6) for 20 min. The sections were treated for 45 min with MRK16 monoclonal antibody, 1:250 dilution in TBS buffer, and then washed in the same buffer containing 0.1% saponine. Incubation for 30 min with anti-mouse IgG antiserum, diluted 1 : 200, was performed and then prediluted streptavidin-biotin complex was applied for 30 min. Samples were stained with diamino- benzidine for 5 min and incubated with 0.5% copper sulfate for intensification of staining. Counterstaining was performed with hematoxylin or Kernecbtrot. Finally, dehydratation, clearing and mounting in Flo-Texx (a synthetic resin, Pittsburg, USA) was performed.

In preliminary experiments, the streptavidin-biotin complex staining procedure with the M RK 16 antibody was performed using samples obtained from human kidney and adrenal gland. Both tissues express high levels of P-glycoprotein, particularly distrib- uted on the surface of epithelial cells of the proximal renal tubules and in cells of the cortex of the adrenal gland (Thiebaut et al. 1987). Using this staining approach, we found a similar distribution of P-glycoprotein in these tissues, compared with peroxidase immu- nohistochemistry used by Thiebaut et al. (1981, data not shown).

Gastric adenocarcinoma specimens were classified according to Laur6n and to WHO International Classification. The subtype of gastric intestinal metaplasia was defined by morphological criteria and by staining the samples with PAS and Alcian Blue (pH 2.5). The sulphated mucopolysaccharides were identified by staining the samples with Alcian Blue at pH 1 according to the method of Lev and Spicer (1964).

Results

Qualitative assessment o f M D R 1 m R N A

Figure 1 shows a N o r t h e r n b lo t h y b r i d i z a t i o n analys is o f the M D R 1 m R N A . The size o f the hybr id ized m R N A c o r r e s p o n d s to a b o u t 4.5 kb , cons is ten t wi th tha t o f the

Fig. l. Northern blot analysis. 10 ~tg of total cellular RNA extracted from non-tumoral gastric mucosa (b, d, f) and gastric carcinoma (a, c, e, g) from untreated patients were separated by electrophoresis and the blots hybridized with the MDR1 3zP-labeled cDNA probe. On the right side, the size (4.5 kb) of the hybridized mRNAs is shown

P-glycoprote in m R N A (Kar tne r et al. 1983). In some samples, the hybr id iza t ion signal could be seen only af ter p ro longed exposure o f the film.

Quantitative assessment of MDR1 mRNA

Figure 2a shows an au to r ad iog raph depict ing the degree of resolut ion ob ta ined by dot -b lo t hybr id iza t ion in non- t umora l gastr ic m u c o s a and ma tched gastric ca r c inoma samples. Figure 2b shows the relative content o f the M D R 1 m R N A s obta ined in the samples studied, expressed as pg o f M D R 1 c D N A bound per 10 I-tg o f total cellular R N A appl ied to the blot. M D R 1 m R N A s were

B

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,< z 0

O~ Q.

0

7

5

3

1

N C

Fig. 2. Relative content of MDR1 mRNAs in gastric carcinoma and non-tumoral gastric mucosa, a Dot-blot analysis: 10 I~g of RNA from non-tumoral gastric mucosa (N) and gastric carcinoma (C) were analyzed as described in Material and methods. On the left side, numbers indicate the matched samples studied. Human kidney (k), hypernephroma (h) were used as positive controls for hybridization, b Quantitative assessment of MDR1 mRNAs expressed as pg of cDNA probe bound per 10 ~tg of RNA. Horizontal lines represent the mean of the MDR1 mRNAs relative level obtained in each group of samples studied

detected in 9 o f 10 non - tumora l gastric mucosae analyzed and in 8 of 10 gastric adenocarc inomas . Similar results were ob ta ined when dot -b lo t was hybridized using 32p-labeled R N A transcripts as a p robe (da ta not shown).

Differences in the relative content o f M D R 1 specific m R N A between these two groups o f samples were not found. Fur the rmore , we observed a significant heterogeneous level o f expression in bo th gastric cancers and non - tumora l gastr ic mucosae . Nevertheless, no correla- t ion was found between the M D R 1 gene expression and the histological type o f adenoca rc inoma , or the degree o f differentiat ion, independent o f the classification used.

Immunohistochemical analysis

The immunoh i s tochemica l local izat ion o f P-g lycopro- tein in n o n - t u m o r a l gastric mucosa f rom one sample analyzed is shown in Fig. 3 a. O f great interest was the finding tha t P-g lycoprote in was only detected, and with a s t rong signal, on epithelial cells o f gastr ic areas with intestinal metaplas ia . Immunoreac t iv i t y to this prote in was not detected in no rma l gastric m u c o s a or gastr ic areas with chronic a t rophic gastritis. Fu r the rmore , the P-glycoprote in expression seemed to be restricted to sul- phomucin-secre t ing type I I I intestinal metap las ia with PAS/Alc ian Blue posit ive staining (Filipe and Jass 1986).

Figure 3 b shows the immunoh i s tochemica l localiza- t ion o f the M D R 1 gene coding p roduc t on a sample f rom a gastric adenoca rc inoma . As can be seen, only some subpopu la t ions o f t u m o r cells express the pro te in provid ing evidence of its he terogeneous dis t r ibut ion in this neoplasm.

Table 1 summar izes the results ob ta ined showing the M R K 1 6 immunoreac t iv i ty and the histological type ac-

Table 1. P-Glycoprotein immunoreactivity and type III intestinal metaplasia in gastric adenocarcinoma and non tumoral mucosa

Sample MRK-16 Metaplasia type Histological types III

N C Laur+n WHO

1 - + + + No Diffuse Signet-ring cell 2 + + + + + Yes Diffuse Signet-ring cell 3 + + + Yes Diffuse Tubular (PD) 4 + + + Yes Intestinal Tubular (PD) 5 + + Yes Diffuse Tubular (PD) 6 + + + - Yes Diffuse Tubular (PD) 7 + + - Yes Intestinal Tubular (PD) 8 + + - Yes Intestinal Solid 9 - - No Intestinal Tubular (PD)

1 0 - - No Intestinal Tubular (PD) 11 - - No Diffuse Tubular (PD) 12 - - No Diffuse Signet-ring cell

The type III intestinal metaplasia was defined by morphological criteria and by staining the samples with PAS/Alcian blue (+ + + ) more than 75% of cells show immunoreactivity; (+ +) 50% of cells showing staining; (+) less than 25% of cells showing staining; ( - ) immunoreactivity non detected. (N) non tumoral gastric mucosa; (C) gastric adenocarcinoma. (PD) poorly differentiated

Fig. 3. P-Glycoprotein expression. a Immunohistochemical analysis of P-Glycoprotein in non-tumoral gastric mucosa. The staining only appears on cells in one focal area of gastric mucosa with intestinal metaplasia subtype III. b Light microscopic appearance of P- Glycoprotein distribution in gastric carcinoma. Some subpopulations of malignant cells express the MDR1 coding product at high level. Hematoxylin and eosin. Magnification, 1 x 400

cording to both Laur6n (1965) and the WHO Classifica- tion (1977). Staining and morphological criteria allowed us to characterize the intestinal metaplasia associated with P-glycoprotein immunoreactivity. This corresponds to the subtype III according to a recent classification proposed (Filipe and Jass 1986). P-glycoprotein was present on malignant cells in 5 of 12 carcinomas studied. In two of these tumors, a strong signal was found with a diffuse pattern of distribution. Correlation between the presence of P-glycoprotein and histological type of tumor, according to both classifications, was not evident.

Discussion

Using the hybridization technique, differences in the relative content of MDR1 mRNAs between gastric carcinomas and non-tumoral gastric mucosae were not evi-

dent in our study. The relative expression of the MDR1 gene in these tumoral samples was lower than the expression found in hypernephroma, considered as the neoplasia with the highest relative content of MDR1 mRNAs (Fojo 1987 a). Additional DNA hybridization studies are necessary in order to define the mechanism of overexpression of this gene in gastric adenocarcinoma.

Detection of MDR1 gene expression in individual cells is essential in those instances in which tumor cell populations are highly heterogeneous, in tumors that are necrotic, or where the tumor has significant stromal elements (Gottesman et al. 1989). In fact, in our experiments not all the RNA samples obtained initially from surgical specimens were hybridized, because some of them showed a significant RNA degradation, probably due to tumoral necrosis.

The immunohistochemical analysis allowed us to define the presence of P-glycoprotein at the cellular level

both in non-tumoral gastric mucosa and carcinoma. The fact that P-glycoprotein was only found on cells of gastric areas with intestinal metaplasia could explain the heterogeneous level of MDR1 RNA transcripts found when total cellular RNA was extracted from non-tumoral gastric mucosa and analyzed by dot-blot hybridization. In addition, the variability in MDR1 mRNAs found in gastric carcinomas by dot-blot hybridization seems clearly to be related to differential expression of P-Glycoprotein in subpopulations of malignant cells.

Gastric mucosa, in contrast to small bowel and large bowel mucosa, has been reported to show a low level of MDR1 expression (Pastan and Gottesman 1987). Our observations are in agreement, in so far as P-glycoprotein was not detected on the surface of normal gastric epithelial cells. In a recent study Robey-Cafferty et al. (1990) showed that P-Glycoprotein is expressed in gastric adenocarcinoma and also in normal gastric mucosa. Their results differ from our own in that they found expression of the gene product in both intestinalized and nonintestinalized gastric mucosa from tumor-bearing stomachs. The differences from our study could be due to the fact that the authors used a different monoclonal antibody (Mab C219) (Gonzalez et al. 1990). This monoclonal antibody also crossreacts with others proteins containing ATP-binding sites (Thiebaut et al. 1989).

The observation that P-glycoprotein was found to be associated only with intestinal metaplasia of gastric epithelium constitutes an interesting finding. However, intestinal metaplasia of gastric epithelium is not a homo- geneous entity. Histochemical and ultrastructural studies have revealed a varied expression of gastric and intestinal phenotype that differ morphologically on mucin se- cretion profiles and enzymatic expression (Filipe and Jass 1986). The heterogeneity of intestinal metaplasia may reflect stages of a dynamic process of injury response in which one form evolves to another or re- gresses. The type III intestinal metaplasia, also called "colonic metaplasia", constitutes a variant of intestinal metaplasia which shows incomplete cell differentiation and atypical phenotypes, sharing features with malignancy and the fetal gut (Filipe and Jass 1986). In addition, some proteins like carcinoembryonic antigen (CEA), mucus-associated antigens (colonic M3C antigen) and human fetal antigens tend to increase gradually in frequency as epithelial changes progress from chronic atrophic gastritis to type III intestinal metaplasia and carcinoma (Filipe and Jass 1986).

Although all the evidence strongly suggests a close relationship between type III intestinal metaplasia and gastric carcinoma, particularly to the "intestinal type", this entity is still not considered as a precancerous lesion. The appearence of P-Glycoprotein in type III intestinal metaplasia of gastric epithelium could be interpreted as a derepression of the MDR1 gene normally silent in human gastric mucosa, that is expressed when cells are differentiated to the intestinal phenotype. In our under- standing, this is the first observation of MDR1 gene overexpression in a human histopathological abnormal- ity characterized by a disorder of cellular differentiation and considered as a precursor of neoplasia (Morson

et al. 1980). Further studies must be carried to define P-glycoprotein expression in gastric dysplasia, a well re- cognised pre-cancerous lesion.

The finding of MDR1 gene expression in the subtype III intestinal metaplasia of gastric epithelium and in gastric carcinoma, may constitute additional evidence sup- porting the hypothesis of an intestinal metaplasia-dysplasia-carcinoma sequence proposed in the histogenesis of some gastric carcinomas. This finding may also indicate that gastric carcinoma cells are capable of differen- tiating towards intestinal epithelia in the same way that regenerating gastric epithelium may differentiate into intestinal metaplasia. In fact, electron microscopic observations (Nevalainen 1986) have confirmed that both the intestinal and diffuse types of gastric carcinoma contain cells that have a number of structural features in com- mon with cells in intestinal metaplasia of the gastric mucosa.

The wide range of P-Glycoprotein expression, detected by an immunohistochemical technique, within some subpopulations of malignant gastric cells is an additional interesting finding. Some gastric carcinomas may contain only a small number of cells expressing MDR1 at a high level. These small populations of cells expressing the protein may be responsible for the acquired resistance to chemotherapy developing during the clinical course of the disease. However, there are no data relating the level of P-Glycoprotein expression for a giv- en malignancy to the ability of the tumor cells to survive a particular course of chemotherapy. The real contribution of this gene and coding product on the mechanism(s) of multidrug resistance in gastric carcinoma re- main to be defined. Our finding encourages further pro- spective clinical studies relating P-Glycoprotein levels with progression of individual gastric carcinomas.

Acknowledgements. The authors thank Dr. Michael Gottesman and Ira Pastan from the Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, USA, for providing the probe 5A of the MDR1 cDNA. Also, we thank Dr. Takashi Tsuruo from the Division of Experimental Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan for providing the MRK16 monoclonal antibody and Dr. Paulina Bull and Dr. Pedro Rosso from the Pontificia Universidad Catblica de Chile for critical review of the manuscript. We thank the Alex- ander von Humboldt Foundation for the donation of the liquid scintillation counter. This work was supported by grant 0413/88 from Fondo Nacional de Ciencia y Tecnologia (Fondecyt), Chile.

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