A comparative study on expression of mucin related antigens in preneoplastic and neoplastic rat colorectal mucosa

ELSEVIER

Cancer Letters

1OS ( 1996) 15-22

A comparative study on expression of mucin related antigens in preneoplastic and neoplastic rat colorectal mucosa Rathindranath Baral*, Dhruba Kumar Sautya, Putul Maity Department

c?fCell Biology, Received

Chittaranjan 2 November

National 1995; revision

Cancer

Institute,

received

37, S.P Mukherjee

30 March

1996; accepted

Road, Crrlcuttu 1 April

700 0X$. indrcr

1996

-----_--Abstract

A polyclonal antibody (PAb35) definedantigen(Ag) was characterizedin associationwith two monoclonalantibody (MAbMl and MAb660) defined mucin Ml and 660 Ags in colorectalmucosaduring 20-methylcholanthrene-induced rat carcinogenesis in the sameorgan.Immunohistochemistry andELBA revealedthat thesethreeantibodieswerereactivewith mostof the colorectalcarcinomas. MAbMl andMAb660 werereactivewith preneoplastic colorectalmucosain rats with no detectablecarcinoma,in contrastto non-reactivitywith PAb35.PAb35reactedwith preneoplastic mucosa,presentadjacentto the canceroustissuesonly. Stainingwasmainly localizedin the cytoplasmof cancercells,gobletcellsand luminalmucous deposits.In control rats,Ml and660 Ags werepresentin gastricmucosa,but not in colorectum.PAb35definedAg wasabsentin gastrointestinalmucosaof controls.ELISA revealed82% reductionin reactivity, whenPAb35 wasreactedwith 2mercaptoethanol (2ME) treatedcolorectalmucosalextracts.Ninety percentreductionwasseenin the caseof MAbMl l-lowever,50% reductionwasdemonstrated whenMAb660 wasreactedwith 2ME treatedextracts. Keywords:

Colorectalcarcinogenesis; 20-Methylcholanthrene;Mucin antigen;Mucosalextract; Preneoplasia; ZMercapto-

ethanol

--.. ._-1__1-

1. Introduction Cancer mucin antigens (Ags) can be utilized as possible diagnostic and therapeutic targets in various human carcinomas (CAs) including colorectal (CR) cancer [ 14,151, a dreaded health problem throughout the world [7]. Some of thesemucin Ags detected and characterized in animal models, have relevance with human CR neoplastic disease [2,10]. Such a rat * Corresponding Nagar, Bhubaneswar

author. Institute of Life 751 007; Orissa, India.

03043835/96/$12.00 0 1996 Eisevier PIf SO304-3835(96)04254-I

Sciences,

Science Ireland

301 Sahid

model of CR CA, induced by 20-methylcholanthrene (MCA) [4,5] has been exploited to find out a cancer associatedAg. In our earlier study, an antigenic glycoprotein with molecular weight (mol. wt.) 35 kDa has been identified in carcinomatous rat colorectal mucosal extracts (CME) but absent in the same of normal rats 161. A polyclonal antibody (PAb35) againstit hasbeenpreparedsuccessfully. This PAb35 is reactive with the serafrom patients having CR CA [6]. Since the immunizing fraction is periodic acidSchiff (PAS) positive and the Ab is reactive with mucin secreting goblet cells of cancerousCR glands,

Ltd. All rights reserved

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the relationship of this Ag with large intestinal mucin moiety cannot be ignored. In this context, the present investigation is designed to characterize this Ag further using PAb35 and to find out its relationship with other known mucin antigenic markers during the process of MCAinduced rat CR carcinogenesis. Monoclonal antibodies (MAbs) against mucin Ag Ml [l] and 660 [8] (markers for 1,Zdimethylhydrazine (DMH) induced rat colonic cancer) have been selected to perform a comparative study with newly described PAb35. This study has demonstrated the expression of PAb35 defined Ag in the neoplastic colorectum, but those defined by MAbMl and MAb660 have expressed both in preneoplasia and neoplasia. 2. Materials

and methods

2.1. Study design

Seventy-two male Sprague-Dawley rats (age, 8 weeks; body weight, 130 g) were taken. The experiment was performed in 2 identical sets, each containing 36 rats. Rats of the first set were utilized for immunohistochemistry (MC). CME was prepared form the second set of rats to perform the enzyme linked immunosorbant assay (ELISA). Each set consisted of 16 experimental, 12 control and 8 normal rats. In each set of experiments, antigenic expression was studied in 4 stages of carcinogenesis, i.e. 8th, 16th, 24th and 32nd week of MCA treatment, involving 9 rats/stage (4 experimental, 3 control and 2 normal). 2.2. Carcinogenesis

CR CA was induced by intrarectal administration of MCA (2 mg MU/week) for 24 and 32 weeks, by the method described earlier [5]. CR mucosa, obtained by MCA treatment at the same dose for 8 and 16 weeks, were taken as preneoplastic mucosa. Control rats received vehicle (benzene) only and normals received no treatment. 2.3. Histopathology

CAs were classified according to Gutmann’s classification [121. Stages I, II and III corresponded to

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CA in situ, submucosa crossing and muscular invasion, respectively, No metastatic CA (Stage IV) was detected within the period of 32 weeks. According to the features described by Decaens et al. [ll], preneoplastic mucosa was characterized by epithelial hyperplasia, polypoid like glands, hyposecretory glands and different grades of dysplasia. These preneoplastic features were observed in rat CR mucosa after 8 and 16 weeks MCA treatment and also in mucosa adjacent to CAs in rats receiving MCA for 24 and 32 weeks. 2.4. Tissue processing

Rats were sacrificed by cardiac puncture under ether anaesthesia and large intestines were opened. Tumors were fixed in 95% ethanol, embedded in paraffin and 4pm sections were cut. Preneoplastic colorectum was coiled up into ‘Swiss rolls’ and processed as Tumours. Different gastrointestinal (GI) organs of control and normal rats were studied in the same way. Serial sections from each sample were prepared. One was stained with hematoxylin-eosin and the other 3 were stained with 3 different Abs. 2.5. Antibodies

MAbMl and MAb660, in the form of lyophilized culture supernatant, were procured as a gift (Professor J. Bara and Dr. C. Decaens, CNRS, IRSC, Cedex, France). Lyophilized powder was dissolved in PBS to its original volume as present before lyophilization. For PAb35, rabbits were immunized with 35 kDa glycoprotein, present in carcinomatous CR mucosa [6]. After serum collection, it was made monospecific by absorption with CR tissues of normal rats, immunoglobulins were precipitated with 50% ammonium sulphate, dialyzed extensively against PBS, concentrated and protein concentration was adjusted by Lowry’s method [ 131. 2.6. IHC An indirect peroxidase method was used, as discussed previously [5]. Briefly, after deparaffinization, tissue sections were incubated with Abs for 1 h. In the case of MAb, culture supematant was used directly and PAb was applied at a protein concentra-

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et al. /Cancer

tion 2Opg/ml PBS. Then antimouse (for MAb) or antirabbit (for PAb) IgG (H + L)-labelled with peroxidase (Diagnostic Pasteur, Marnes La Coquette, France) was applied at a l/100 dilution for 45 min. Finally peroxidase activity was revealed using 0.06% aminoethylcarbazol and 0.15% H202. Sections were counterstained with hematoxylin. A preneoplastic or neoplastic gland was considered positive when it contained at least one positive goblet cell. A zone of carcinomatous mucosa with complete loss of differentiation was evaluated as positive if it contained at least 3 positive cells. The specificity of staining was controlled by different absorption as described by Decaens et al. [8]. Sections known to stain positively (gastric mucosa or CR adeno CA) were included in each run, received either primary Ab or non-immune serum (mouse or rabbit), as positive and negative controls, respectively. 2.7. CME preparation

CME were prepared from rats of an identical set of experiments. Mucosa of rats were scraped from colorectum and suspended in cold 50 mM Tris-HCl, with 0.15 M NaCl (pH 7.4) containing 2 mM PMSF, 2 mM EDTA, 0.02% sodium azide and homogenized separately. Homogenates were centrifuged successively at 6000 and 20 000 rev./min for 30 min in each case, according to the method described [3]. Supernatants were used as CME after protein estimation by Lowry’s method [13]. 2.8. 2Mercaptoethanol(2ME)

reduction

2ME (0.2 M) was added to the CME for 5 h at 37°C after protease inhibition with 1 mM PMSF. Then it was alkylated using 1 M iodoacetamide for 16 h in the dark [8]. The reduced Ag was then dialyzed and concentration was adjusted .

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dase activity was revealed using 0.075% 2,2’azinobis (3-ethylbenzthiozdi~~I~onic acid) and 0.09% H,Oz. The absorbance was measured at 410 nm. 3. Res&s 3.2. Detection of Ags by IHC

3.1. I. Staining of CR Ch Total number of 59 C4s w@re~1~~ and rectum of 8 r and 32 weeks. As

.in the colon

from CA in situ (stalge I) ED lar layer (stqe III); 98, 88 tive with PAb35, MAb&l tively. Staining with 3 Abs was lac&& in the cytoplasm of cancer cells @g. I), mucous deposits and in the lumen of glands. 3.1.2. Staining of non-neoplastic CR mucosa

and preneoplastic

Non-neoplastic and preneoplastic features were present in CR mucosa of rats treated with MCA for 8 and 16 weeks and these mucosa were studied for the expression of 3 types of Ags. MAbMl and MAb660 were reacted with hyperplastic (Fig. 2) and dysplastic (Fig. 3) mucosal @a&s and staining was mainly localized in glandular epithelial cells and/or mucous deposits. Reactivity of these 2 MAbs was also detected in the gob-let cells of glands, with no detectable histological modifications (Fig. 4). At the 8th week, the total number of 67 glands with MAbMl reactivity and 72 glands with MAb660 reactivity were counted in CR mucosa of 4 rats. At the 16th week, 79 Table 1 lmmott&jsto&amical sea&ivities of antibodies with induced cohic and rectal cctrcinomas

MCA

2.9. ELISA Antibodies

CME (lO@g/ml) prepared from each rat, were coated separately on microtitre plates (Nunc Immunoplates, Denmark) by overnight incubation at 4”C, with and without 2ME reduction. Plates were incubated with primary and secondary Abs at the same concentration as used in IHC. Finally, peroxi-

No. of positive carcinomas

Positive carcinomas/ total

Stage I Stage II Stage III PAb35 MAbMl MAb660

18 17 18

21 20 19

19 15 16

Ill?.

OF

carcinomas studied (S) 58/59 (98) SZ/SY(88) s3m (90) --

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with PAb35. Characteristics of preneoplastic mucosa were also seen in the region adjacent to CAs. These preneoplastic tissues were often reactive with PAb35. At the 24th week, 87, 121 and 156 glands were positive with PAb35, MAbMl and MAb660, respectively, counted in the CR mucosa of 4 rats and at 32nd week, 101, 135 and 163 glands positive with these 3 Abs were counted in the mucosa of same number of rats.

3.1.3. Staining of normal GI mucosa

Fig. 1. A poorly differentiated carcinomatous mucosa of rat, treated with MCA for 32 weeks. Cytoplasm of few cancer cells showed positive staining with PAb35. Immunoperoxidase, X300.

and 91 glands were counted, reactive with MAbMl and MAb660, respectively. All these non-neoplastic and preneoplastic glands appeared to be negative

Pig. 2. Hyperplastic MAb660 reactivity.

glands in the rectal mucosa of a rate treated Immunoperoxidase, x480.

CR mucosa of control and normal rats, sacrificed at the end of the 8, 16,24 and 32 weeks, showed absence of staining (Fig. 5) with 3 Abs. Besides colorectum, the Ml and 660 Ags were expressed in the mucous cells of surface gastric epithelium (Fig. 6). Few duodenal goblet cells were also positive with MAb660. Normal GI tracts were negative with PAb35.

3.2. Detection of Ags by ELBA CME, with or without 2ME reduction, were reacted with PAb35, MAbMl and MAb660 (Table 2). Reactivity of PAb35 with CME prepared from 8 and

for 16 weeks with MCA.

Luminal

mucous

deposit of one gland is showing

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Fig. 3 MAbM 1 positive dysplastic glands in the colonic mucosa of a rat treated with MCA for 24 weeks. Jmmunoperoxidasr. x480.

16 weekLS MCA treated rats was negligible, but promine:nt reaction was observed with extracts of rats receivin g treatment for 24 and 32 weeks. This reaction wsts reduced significantly (82%) after 2ME treatmetIt. CME, prepared from rats of 4 stages,were

reacted with MAbMl and MAb660. Following 2PVfE reduction, recognition of Ags in extracts was remarkably reduced (90%) in the case of MI. HaIWever, reduction was 50% during reaction wfith MAb660.

Fig. 4. Histologically normal colonic mucosal glands of a rat treated with MCA for 8 weeks, showing reactivity with MAblrhlI Jmmunoperoxidase, x 480.

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Fig. 5. Normal distal colonic mucosa of a control rat, showing no reactivity with PAb3.5. Immunoperoxidase, x300.

4. Discussion Immunohistochemical study on the expression of PAb35 defined Ag with reference to the AgMl and Ag660 during the process of CR carcinogenesisrevealed that PAb35 reacted with 98% of the CR CA;

88 and 90% CAs were positive with MAbMl and MAb660, respectively (Table 1). Decaens et al. reported 68% AgMl [9] and 100% Ag660 [8] positivity in colonic CAs induced in Wistar rats by DMH. Preneoplasticmucosaappearedeither in the colorecturn of 8 and 16 weeks MCA treated rats or in the

Fig. 6. Surface gastric epithelium of a control rat, reactive with MAbMl . Immunoperoxidase, X300.

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Table 2 Immune reactivities in rat colorectaI mucosal extracts during carcinogenesis, detected by ELISA Antibodies

Weeks of MCA treatment 8

PAb35 (82%) MAbMl (90%) MAW60 (50%)

16

24

32

-2ME

+2ME

-2ME

+2ME

-2ME

+2ME

-2ME

+2ME

N

N

N

N

0.146 20.03 1 0.151 +0.020

0.016 *0.005 0.078 kO.013

0.159 jzO.029 0.161 *0.012

0.016 *0.002 0.077 +0.008

0.164 + 0.025* 0.192 rto.03 1 0.172 Yko.015

0.03 1 +0.005 0.019 *0.003 0.084 -+0.012

0.186 kO.032 0.213 ti.037 0.178 *0.025

0.03 1 rto.003 0.021 *O.OOS 0,090 *0.017

Figures in parentheses indicate percent of reduction of immune reactivities after 2ME treatment, -2ME and +2ME, before and after 2ME reduction, respectively. N, negligible. *Mean values of 4 different observations (i standard deviation) at 410 run are presented in each case.

region adjacent to the CAs in rats receiving 24 and 32 weeks MCA treatment. Both of these preneoplastic mucosa were reactive with MAbMl and MAb660. This observation is in agreement with previous reports [ 1$1. However, preneoplastic mucosa adjacent to the CAs were only reactive with PAb35. Using ELISA the investigation was extended to confirm the association of Ags with preneoplastic and/or neoplastic processes. MAbMl and MAb660 were reacted with CME at 4 stages of CR carcinogenesis and Ag concentrations were linearly increased with progression of the disease (Table 2). However, PAb35 was only reacted with the CME prepared from CA bearing rats, confirming its association only with cancer. For further characterization, CME were treated with 2ME and then, reacted with 3 different Abs by ELISA. In all cases, concentrations of reactive Ags were reduced following 2ME treatment. However, the extent of reduction was not identical. In the case of PAb35, 82% reduction was observed. MAbMl reactivity was reduced to 90% and this observation correlates well with the data reported by Bara et al. [2]. They concluded that the reactivity of these Ags with MAbMl are dependent on their conformation, stabilized by disulphide bridges. On the contrary, 50% reduction was noted in the case of MAb660. The preservation of MAb660 reactivity after 2ME treatment was demonstrated previously by Decaens et al. [S] and they suggested that, unlike the Ag Ml, 660 Ags are not conformational. Therefore, with re-

spect to the 2ME sensitivity, Ag recognized by PAb35 is closer to the Ml than Ag660. In conclusion, it can be stated that MAbMl and MAb660 defined Ags have significance in early carcinogenesis at rat colorectum, but PAb35 selectively reacts with the carcinomatous colorectum only. Besides its cross reactivity with the serum from CR cancer patients [6], the role of PAb35 defined Ag in human large intestinal neoplasia should he elaborated further. Acknowledgements

The authors thank Decaens, France, for bodies. The financial Council of Medical acknowledged.

Professor J. Bara and Dr. C. their gift of monoclonal antiassistance given by the Indian Research, New Delhi, is also

References [ 1J Bara, J., Gautier, R., Daher, N., Zagbouani, H. and Decaens, C. (1986) Monoclonal antibodies against oncofetal mucin Ml antigens associated with precancerous colonic mucosa. Cancer Res., 46.3983-3989. [2] Bara, J., Gamier, R., Mouradian, P., Decaens, C. and Daher, N. (1991) Oncofoetal mucin Ml epitope family: characterization and expression during colonic carcinogenesis. Int. J. cancer, 47,304-310. [3] Bagchi, S. and Das, K.M. (1984) Detection and partial characterization of Cmhn’s disease tissue specific proteins retognized by Crohn’s disease sera. Clin. Exp. lmmunol., 55, 41-48.

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[9]

R. Bum1 et al. / Cuncer Baral, R.N., Chakraborty, A. and Maity, P. (1993) Alterations in large gut associated lymphatic tissues (LGALT) during experimental colorectal carcinogenesis. lnd. J. Exp. Biol., 3 I, 793-796. Baral, R.N. and Maity, P. (1992) Induction of colorectal cancer in rats by 20.methylcholanthrene. Cancer Lett., 61, 177-183. Baral, R.N. and Maity, P. (1996) Detection and partial characterization of a 35 kDa antigenic glycopeptide in relation to the experimental colorectal carcinoma. J. Exp. Clin. Cancer Res., in press. Boring, CC., Squires, T.S. and Tong, T. (1994) Cancer statistics. C.A. Cancer J. Clin., 44.7-26. Decaens, C., Gamier, R., Bara, J., Daher, N., Pendu, J. and Burtin, P. (1988) A new mucin associated oncofetai antigen, a marker of early carcinogenesis in rat colon. Cancer Res., 48, 1571-1577. Decaens, C., Nardelli, J., Bara, J. and Burtin, P. (1984) Colonic and gastric mucus-associated antigens: a comparative immunohistological study in precancerous and cancerous rat intestinal mucosa. Eur. J. Cancer Clin. Oncol., 20, 975-981.

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