Early Stages of 1,2-Dimethylliydrazine-Induced Colon Carcinogenesis Suppress Immune-Regulated Ion Transport of Mouse Distal Colon1

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[CANCER RESEARCH54, 5930â€”5936,November15, 19941 Early Stages of 1,2-Dimethylliydrazine-induced Colon Carcinogenesis Suppress Immune-regulated Ion Transport of Mouse Distal Colon1

Russell R. Broaddus, Michael J. Wargovich, and Gilbert A. Castro2

Departments of Physiology and Cell Biology (R. R. B., G. A. C.] and Medical Oncology [M. J. WI, University of Texas Medical School [R. R. B., G. A. C.] and University of Texas M. D. Anderson Cancer Center [M. .1. WI, Houston, Texas 77225

ABSTRACT then to adenocarcinoma. In addition to these genetic events, epige netic and microenvironmental changes also contribute to the devel Genetic events, such as gene deletion, mutation, and amplification, are opment of neoplasia (3). For colon cancer, these latter changes have involved in the development and progression of colonic neoplasia The importance of interactions between immune cells and neoplastlc cells in been much less intensively studied. influencing the pathogenesis of colon cancer is suggested by the clinical Compelling results suggest that local mucosal immunity is one such efficacy of levamisole, an immunostimulant, in the treatment of colon microenvironmental factor important in the pathogenesis of colorectal cancer and by the association of local lymphocytic infiltration with Im carcinoma. Colon adenocarcinomas often contain an inflammatory proved prognosis. Thus, the immune cell-neoplastic cell interaction can be infiltrate, typically consisting of T lymphocytes with some macro viewed as being analogous to various host-parasite relationships that phages and B lymphocytes (4). The presence of lymphocytic infiltra involve the immune system attempting to contain the intruding parasite tion or of lymphoid follicles near resected colon cancers is associated on the one hand and the parasite attempting to escape detection and with an increased patient survival rate (5, 6). Levamisole, a nonspe rejection on the other. This study is an attempt to gain a better under standing ofsuch interactions by examining possible effects ofa developing cific stimulator of many different arms of the immune system, in tumor on the immune system. Colon cancer was experimentally induced in combination with 5-fluorouracil significantly improves disease-free mice to examine potential effects of carcinogenesis in the colon on local survival and overall survival in patients with Stage C resected colon mucosal immune function in situ, using the expression of type I hyper cancer (7). These observations underscore a delicate modulatory role sensitivity as an index of immune function. Antigen-induced changes in of the immune system, especially the mucosal immune system of the net ion transport, a quantifiable correlate of type I hypersensitivity, were colon, in colonic neoplasia. To enhance an understanding of the measured in the colon of mice following the administration of the procar interaction between the mucosal immune system and neoplastic tissue, cinogen 1,2-dimethyihydrazine (DM11).Segments of colon and small in we investigated the effects of experimental colon carcinogenesis on testine from mice immunized by infection with Trichinella spiralir secrete immunity in the colon and small intestine. ions, manifested as a rise in transmural short-circuit current, when dial lenged with T. spiralis antigen in Ussing chambers. Antigen-induced rise in Systemic administration of the procarcinogen DMH3 to CF-i mice transmural short-circuit current Is mast cell dependent and occurs only in is a well-established laboratory model of colon carcinogenesis. DMH immunized mice. To determine the effects of early stages of colon card selectively induces colonic adenomas and adenocarcinomas (8, 9) nogenesis on this mucosal immune response, mice were immunized with T. while only rarely causing liver or small intestine tumors (10). Admin spiralis prior to and after 6 weekly injections of DM11. Responsiveness to istration of DMH causes a continuum of morphological changes from antigenic challenge was suppressed in the distal colon 4â€”6weeks after the normal colonic epithelium to carcinoma. One injection of DMH final injection of DMH. Although responsiveness to antigen was sup causes the development of aberrant crypts within 2 weeks (11), while pruned, the colonic epithelium remained sensitive to direct stimulation by 6 injections of DMH, 1 per week for 6 weeks, typically causes grossly exogenous secretagogues. Decreased antigen responsiveness observed In visible adenomas and carcinomas of the colon within 4â€”6months (8, the distal colon was not due to generalized Immunesuppression by DM11, because the proximal colon and thejejunum retained their responsiveness 9). Within the same time frame as the histological development of to antigen, and immune rejection of a secondary T.spiralis infection from aberrant crypts, there is electrophysiological evidence of inhibition of the small Intestinewas not altered. Visible tumors eventually developed 30 the Na@-K@-ATPase(12) and stimulation of Na@-H@exchange (13) in weeks after the final Injection of DM11 only in the distal portions of the the distal colon. These changes in ion transport are important, because colon. These results suggest that early stages of DM11-induced carcino Na@influx into the cell (14) and increased intracellular pH (15) are genesis are associated with the suppression of mucosal immune function, early events in mitogenesis and may, at least partially, be the basis for as measured by Immune-regulatedion secretion, in the distal colon but not the hyperproliferation observed in aberrant crypts (16). in the proximal colon or jejunum. Future eluddation of the mechanisms The hyperproliferative lesions, aberrant crypts, adenomas, and ad by which this localized hnmunosuppression occurs may provide clues to enocarcinomas of the colon induced by DMH are biologically and the microenvironmental changes necessary for tumor progression in the histologically quite similar to those seen in humans (17). Importantly, distal colon. there is pathological evidence that DMH-induced colon tumors (18) resemble human tumors (2) in that both follow an adenoma-to INTRODUCTION carcinoma sequence. Because of the potential progression of early Recent molecular biological evidence (1) supports histological ev changes to malignancy, the study of the premalignant hyperprolifera idence (2) indicating an adenoma-to-carcinoma sequence in the de tive lesions and aberrant crypts is crucial in understanding the patho velopment of colon cancer. The accumulation of mutations in onco genesis of colon cancer. genes and deletions and mutations of tumor suppressor genes allows Immunity in the small intestine and colon of DMH-treated mice for the progression of hyperproliferative epithelium to adenoma and was measured by quantifying a vital physiological function, ion trans port, that is regulated by the mucosal immune system (19). Immune Received 6/14/94; accepted 9/20/94. regulated intestinal ion transport has been described for a variety of The costs of publication of this article were defrayed in part by the payment of page host-antigen systems. Antigenic challenge of intestinal segments from charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NIH Grant P01-DK37260 and the University of 3 The abbreviations used are: DMH, 1,2-dimethylhydrazine; PGE@, prostaglandin E@; Texas-Houston Health Science Center Mucosal Biology Program. 5-rn,,5-hydroxytryptamine;L@short-circuitcurrent;KRBbuffer,Krebs-Ringerbicar 2 To whom requests for reprints should be addressed, at Department of Physiology and bonate buffer; TGF-@, transforming growth factor @;cAMP, cyclic AMP; cGMP, cyclic Cell Biology, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225. GMP. 5930

laboratory rodents sensitized to dietary proteins (20, 21) or parasites Segments of intestine were allowed to stabilize for 30 mm after @ (2Z 23) resultsin an immediate(typeI) hypersensitivityreaction, mounting in the chambers to obtain a steady-state [email protected] basal is expressed as net epitheial anion secretion. In Ussing chambers, net an index of net active ion transport across the serosal and mucosal anion secretion is manifested electrophysiologically as a rise in trans surfaces. The tissues were stimulated with antigen or secretagogues mural I.e. Applying similar principles, we have recently described a added to the serosal bathing fluid. T. spiralis antigen was added at a method to quantify colonic mucosal immune function in situ (24). concentration of 50 ,.agprotein/mi of Ussing chamber fluid. PGE@and Briefly, segments ofcolon from mice immunized by infection with the 5-HT were added at final chamber concentrations of iO@ M and 10@ intestinal nematode parasite Trichinella spiralis undergo an increase M, respectively. After addition of antigen or secretagogue, the maxi in Inc (biI,,@)after challenge with T. spiralis-derived antigen in Ussing mum change in I@ (@M@)from the basal state was calculated and chambers. The antigen-induced AL@is inhibited by furosemide, indi expressed as @ eating that the is most likely due to net Cl@secretion by the colon. T.spiralisantigenbridges1gBboundto mucosalmastcells,triggering Histology the release of anaphylactic mediators that stimulate epitheial ion secretion. Ion secretion and the resulting colomc i@L@thereforeserve Colomc segments from tumor-bearing mice were slit longitudinally as sensitive indicators of local immune function. In this study, anti and rinsed free of luminal contents with ice-cold KRB buffer. The gen-stimulated changes in colonic lac in DMH-treated mice were used segments were fixed in 10% buffered formalin, embedded in paraffin, as a physiological correlate of mucosal immunity to investigate the and cut into [email protected]. Sections were stained with hematoxylin impact of early stages of colon carcinogenesis on the local immune and eosin before examination by light microscopy. system. Experimental Design MATERIALS AND METHODS Ussing Chamber Studies. To investigate the effects of early Anisnala stages of DMH-induced colon carcinogenesis on the development of mucosal immune responsiveness, 6 weekly injections of DMH were Male CF-i mice (Harlan Sprague-Dawley, Houston, TX), 22â€”24g superimposed on a primary T. spiralis infection. Group 1 mice were initial body weight, were used as experimental hosts. The mice were infected with T. spiralis 1 week after the first DMH injection and then housed in cages with standard bedding and were given a laboratory given the remaining 5 weekly DMH injections. I@ responses to T. pelleted formula diet and tap water ad libitum. On the day of carcin spiralis antigen and secretagogues were then examined 8 weeks after ogen administration, DMH was dissolved in 1 m@ssodium EDTA and the primary infection (4 weeks after the sixth injection of DM11). the pH was adjusted to 6.5 with 1 N NaOH. Mice were given i.p. Infected mice given 6 weekly injections of EDTA carrier solvent injections of 20 mg@g of DM11, housed in microisolators (Lab Prod served as immune controls. ucts Inc., Maywood, NJ) under a chemical fume hood for 6 h, and then To determine the effects of early stages of DMH-induced colon returned to standard cages in the Animal Care Facility. carcinogenesis on an established mucosal immune response, group 2 Mice were immunized by infection with 400 muscle stage larvae of T. spirali&The numberof intestinalwormsestablishedfollowinga mice were given a primary T. spiralis infection, allowed 8 weeks to develop full inununity, and then given 6 weekly injections of DMH. primary or challenge infection with T. spiralis was quantified accord lac responses to stimulation with T. spiralis antigen and secretagogues ing to established methods (25). Infective larvae were collected from the skeletal muscle of parasitized mice according to the method of were examined 6 weeks after the sixth injection of DM11. Immune Castro and Fairbairn (26). T. spiralis antigen was prepared from mice receiving 6 weekly injections of EDTA served as controls. infective larvae (27), analyzed for protein (28), and stored in aliquots Worm Rejection Studies. To determine the effects of early stages at â€”20Â°C. of DMH-induced colon carcinogenesis on functional immunity, worm rejection from the small intestine was measured. Groups of mice were Ion Transport Measurements immunized with a primary T. spiralis infection of 400 larvae after the second (group 3) or sixth (group 4) DM11 injection. The effects of one Antigen-induced and secretagogue-induced ion transport in the injection of DMH were examined by administering DMH either 1 day jejunum and colon were measured in Ussing chambers (23) 8 weeks before the primary infection (group 5) or 1 day before the secondary after mice were given the primary T. spiralis infection. Mice were challenge infection (group 6). For all 4 groups, a challenge infection killed by cervical dislocation, and the small intestine and colon were of 400 larvae was given 30 days after the primary infection. The surgically removed and rinsed free of luminal contents with KRB number of worms that established in the small intestine was deter buffer (pH 7.4) as described previously (23). The jejunum and colon mined 2 days after the challenge infection (25). were slit longitudinally, placed in ice-cold KRB buffer, and gassed with a mixture of 95% 02-5% CO2 until the tissues were mounted in Materials Ussing chambers. The colon was divided into four segments, desig nated near-proximal, mid-proximal, mid-distal, and far-distal, begin POE2 and 5-HT were purchased from Sigma (St. Louis, MO). ning at the cecum and extending distally to the anus. Jejunal segments DMH was purchased from Aldrich (Milwaukee, WI). PGE@was and the four colonic segments were mounted as full-thickness, flat solubiized in ethanol and 5-lU was solubiized in H20. sheets between two Ussing half-chambers with an aperture of 0.516 @2 Each segment was bathed on the mucosal and serosal surfaces Statistics with 10 ml KRB buffer gassed with 95% 02-5% CO2 and maintained at 37Â°C.Colonic and jejunal segments were voltage clamped at zero Student's t test was used to compare the means of two experimental trausmural potential difference using a VCC-600 voltage current groups (29). When more than two means were compared, a single clamp (Physiologic Instruments, San Diego, CA). A continuous re analysis of variance was used, and the Duncan test was used to @ cord of with respect to time was recorded on a BD-41 Kipp Zonen analyze multiple comparisons between the means (29). The results are recorder (Delf@ the Netherlands). expressed as mean Â±SE. P = 0.05 or less was considered significant. 5931

RESULTS A. B. 250 Segments of mid-distal and far-distal colon from group 1 mice (6 -I-- weekly DMH injections superimposed on a primary T. spiralis infec 200 tion) demonstrated decreased responsiveness to T. spiralis antigen 150 (Fig. 1A). However, DMH treatment did not significantly affect an tigen-induced I,@responses in the proximal colon or jejunum (Fig. 1, 100 B and C). Collectively, these results suggest that early stages of DMH-induced colon carcinogenesis suppress mast cell-dependent im 5: 12_@ mune reactions of the distal colon but not of the proximal colon or jejunum. PGE2 5-HT @ Inhibition of antigen4nduced in the distal colon could be due Fig. 2. Responsiveness of distal colon to exogenous Cl secretagogues. Segments of to either a breakdown in local mucosal immune reactions or the distalcolonfrommiceimmunizedbyinfectionwithT.spiralisafterthefirstof 6 weekly injections of DMH or EDTA carrier solvent were challenged in Ussing chambers with inability of the colonic epithelium to secrete anions after receiving POE2 (A) or s-rn' (B). Columns, mean; bars, SE; n. number at base of column. signals generated by immune cells. The basal I,@of the distal colon

from T. spiralis-infected mice (19.1 Â±3.8 p.A/cm2, n = 7) was not A. @o Distal Colon significantly different from the basal I,@of group 1 DMH-treated mice (27.1 Â±4.8 @.aA/cm2,n= 6). Therefore, the sensitivity of the epithe @c-40 0 immunecontrol hum to physiological stimulation was determined by measuring the E @ evoked by the addition of exogenous PGE@and 5-HT, which are @30 J_ . both mast cell-derived mediators and epithelial C1 secretagogues. These secretagogues were chosen because we have previously shown j20 that POE2 and 5-HT are important mediators of the colonic AI@ in response to T. spiralis antigen stimulation (24). DMH treatment had @ 10 no significant effect on epithelial responsiveness to either PGE@ or 5-HT (Fig. 2), suggesting that the epithelium retains the ability to 14 10 A â€” respond to immune agonists. The epithelial I,@responses to PGE@and 5-HT indicate that the Mid-Distal Far-Distal @ decreased antigen-induced observed in the distal colon is sec ondary to a defect in the immune processes that contribute to the B. formation of a local type I hypersensitivity reaction. Such processes 25 Proximal Colon include antigen presentation by macrophages, B cell production of IgE, T cell secretion of cytokines, and mast cell release of chemical 04 20 mediators. Because in group 1 the DMH injections were occurring as the host was mounting a primary immune response to T. spiralis, this 15 defect could involve any of these components necessary for the expression of type I hypersensitivity. To facilitate the identification of the immune defect, group 2 mice were immunized with a primary T. 10 spiralis infection and then 8 weeks postinfection given 6 weekly injections of DMH. With this protocol antigen presentation, T cell and I 5 B cell activation, and 1gB production have all occurred before the onset of DMH injections. The group 2 protocol therefore examines the effect of DMH treatment on mast cell communication with the colonic Near-Proximal Mid-Proximal epithelium. Distal and mid-proximal colon I@ responses to T. spiralis antigen C. were decreased in group 2 mice (Fig. 3, A and B). However, antigen 80 Jejunum induced AI,@in the near-proximal colonic segment and the jejunum 70 was unaffected by DMH treatment (Fig. 3, B and C). Therefore, as in 60 group 1 mice, early stages of DMH-induced colon carcinogenesis resulted in suppressed antigen-stimulated I,@responses in the more 50 distal portions of the colon, but antigen responsiveness in the proximal 40 colon andjejunum remained intact. As in group 1, the colonic basal I@ 30 and the i@I@elicited by exogenous Cl secretagogues were not inhib ited by DMH (results not shown). These observations suggest that the 20 defect in antigen responsiveness in the distal colon is in mast cell 10 functioning or in the transductive pathway connecting the mast cell I 8 ______derivedsignalstothecolonicepithelium. A group of DMH-treated mice was examined for tumors 30 weeks Fig. 1. Antigen-induced change in short-circuit current (@M,@)inthe distal colon (A), proximal colon (B), and jejunum (C). Segments of colon and jejunum from mice after the final dose of DMH (Fig. 4). Normal colonic mucosa is immunized by infection with T. spiralis after the first of 6 weekly injections of DMH or arranged in orderly crypts containing differentiated goblet cells EDTA carriersolvent were challenged in Ussing chamberswith T. spiralis antigen. packed with mucin (Fig. 4A). Adenomas were characterized by crypts Columns, mean; bars, SE; n, number at base of column. Asterisk, significant difference (P < 0.05) as compared with respective immune control. with an increased number of mitotic figures and a decreased number 5932

A. colon requires a carcinogenic progression rather than acute DNA Distal Colon damage following one injection of DMH. 0 immuneControl Worm rejection experiments were performed to examine the effect of DMH treatment on a functional immune response. Mice develop U DMH-treated I immunity to T. spiralis such that a secondary challenge infection is expelled from the small intestine more rapidly than a primary infec tion (32). Despite several different DMH injection schedules, there was no difference between immune, DMH-treated mice and untreated I * immune mice in their ability to reject a secondary infection (Table 1). These results indicate that while DMH treatment causes suppression @ of T. spiralis antigen-induced in the distal colon, it does not Far-Distal Mid-Distal inhibit a functional immune response (worm rejection) occurring in B. the small intestine. Proximal Colon DISCUSSION I DMH treatment during a primary T. spiralis infection (group 1) @ I resulted in suppressed antigen-induced in the distal portions of the colon (Fig. IA). The suppressive effect was also observed in mice immunized prior to the onset of DMH injections (group 2) such that g mast cells bearing surface IgE were present in the mucosa before the onset of carcinogenesis (Fig. 3A). Previously, it has been reported that

11 DMH administration inhibits Naâ€•-K@-ATPase(12) and stimulates Na@-H@exchange (13) in the distal colon. Despite these changes in Near-Proximal Mid-Proximal membrane transport, the epithelium of the distal colon maintained normal responsiveness to exogenous Cl secretagogues (Fig. 2), sug C. gesting that carcinogenesis in the distal colon disrupted mast cell 100 __I__ communication with a normally secreting epitheium. There are several lines of evidence that the inhibited antigen @8o induced @M5@inthe distal colon was not due to systemic immunosup pression by the carcinogen: (a) antigen-stimulated L@responses of the more proximal regions of the colon (Figs. lB and 3B) and of the jejunum (Figs. 1C and 3C) were not decreased by DMH treatment; (b) a functional immune response, worm rejection from the small intes tine, was not affected by DMH (Table 1). Expulsion of primary and

12 secondary T. spiralis infections is delayed in other experimental G models of immunosuppression, such as in athymic mu/mu rats (33), ImmuneControl DMH-Treated athymic nude mice (34), W/Wv mast cell-deficient mice (35), mice pi&3.Antigen-inducedchangeinAL@inthedistalcolon(A),proximalcolon(B),and exposed to sublethal irradiation (36), and mice treated with cortisone jcjunum (C). Segments of colon and jejunum from mice given six injections of DMH or (37). Furthermore, in studies of intestinal immune-regulated ion trans @ EDTA carrier solvent 8 weeks after a primary infection with T. spiralis were challenged port, antigen-induced is absent in athymic mu/mu rats (38) and in Usaingchainbers with T. spiralis antigen. Columns. mean; bar@SE; n, number at base ofcolwnn. AsterLch; significant difference(P < 0.05)as compared with respective immune 70%reducedinmastcell-deficientW/Wvmice(39).IntactI@re control. sponses in the proximal colon and jejunum and intact immune expul sion of a secondary infection all suggest that the suppressive effects of DMH-induced carcinogenesis on mucosal immunity are not general of mucin-containing goblet cells. Some adenomas were severely dys lied but instead are localized to the distal colon. plastic, containing nests of anaplastic cells without the normal crypt The dichotomy between the distal colon and the proximal colon architecture (Fig. 4B). Adenomas developed only in the distal portions regarding antigen responsiveness is also observed in other biological of the colon of DMH-treatedmice;no tumorsdevelopedin the aspects of both DMH-induced and human colon tumors. For example, proximal colon or small intestine. Therefore, the suppression of anti DMH treatment causes tumors predominantly in the distal, rather than gen responsiveness observed in group 1 and 2 mice occurred only in the proximal, colon (17). In the present study, DMH-treated mice the distal colon, the portion of the colon that eventually developed allowed to survive for 30 weeks developed adenomas only in the adenomas. distal portions of the colon (Fig. 5); no tumors were observed in the A possible carcinogenic mechanism of DMH is methylation of proximal colon. Furthermore, the tumors induced in the distal colon DNA (30). Maximum DNA damage in the colon occurs 6-12 h after are histologically different than those in the pcoximal colon (40). one injection of DMH, with repair occurring over the next 72 h (31). Following injection of DMH there is inhibition of the Na@â€•-Kâ€•-A1Tase To determine if one acute exposure to DMH is sufficient to inhibit (12) and stimulation of Na@-H@exchange (13) in the distal, but not responsiveness to antigen, mice were infected with T. spiralis, al proximal, colon. These observations provide strong evidence that the lowed 8 weeks for full immunity to develop, and then treated with one distal portion of the colon is more susceptible to the carcinogenic @ injection of DMH. Colonic in response to antigen, evaluated mechanism of DMH. 6-12 h post-DMHinjection,wasnotdecreasedcomparedwithim Human colon carcinomas also exhibit a proximal-distal dichotomy. mune controls receiving one injection of EDTA carrier solvent (Fig. Allelic deletions of chromosomes l'7p, 18, and Sq are all more @ 5).Thissuggeststhatsuppressionofantigen-induced inthedistal frequent in distal tumors than in proximal tumors (41, 42). Increased 5933

COLON CARCINOGENE5I5 SUPPRESSES MUCO@ALIMMUNE FUNCI'ION

Fig. 4. Histological sections of distal colon from control (A) and DMH-treated (B) mice. DMH treatment causes an increase in muco sal thickness and colon circumference due to crypt hyperplasia. A, normal colonic mucosa with differentiated, mucin-containing goblet cells in characteristic crypts; B, section through an adenoma, 30 weeks after the final dose of DMH. The tumor contains prominent ::. cysts, crypts with goblet cells, less-differentiated crypts lacking gob p@ let cells, and nests of anaplastic cells. Adenomas were not observed in the proximal colon of DMH-treated mice. H & E, X 200. 4

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experimental rodent studies of colorectal carcinoma suggest that there Colon 0 Immune is a biological difference between proximally and distally located tumors. Furthermore, the initiation and progression of proximal and . DMH-treated distal colon cancers may involve different genetic mechanisms. It is possible that the differences in the biology and genetic mechanisms of distal versus proximal colon tumors are the basis for the suppression of antigen-induced ion transport in the distal colon. One exposure to DMH, which is known to be sufficient to cause DNA methylation (30) and subsequent DNA repair (31), is not asso dated with suppression of antigen responsiveness in the distal colon @ 10 (Fig. 5). However, decreased antigen-induced responses in the distal colon (Figs. IA and 3A) are temporally associated with previous reports of Na@-Kâ€•-ATPaseinhibition(12), Na@-H@exchange stimu Far-Distal Mid-Distal lation (13), and the histological appearance of aberrant crypts (1 1) in Fig. 5. Effect of 1 DMH injection on antigen-induced @I,,inthe distal colon. Mice DMH-treated mice. Furthermore, the observed suppression of colonic were immunizedby infection with T. spiralis and 8 weeks latergiven one injectionof DMH or EDTA carrier solvent. Segments of distal colon were challenged in Ussing mucosal immune function (Figs. 1 and 3), the changes in epithelial chamberswithT.spiralisantigen6â€”12haftertheDMHIEDTAinjection.Columns,mean; membrane transport (12, 13), and the development of aberrant crypts bars, SE; n, number at base of column. (45) all preferentially occur in the distal, rather than the proximal, c-myc expression is correlated with cancers in the distal colon (43). colon. Altered Na@-K@-ATPase(14) and Na@-W exchange (15) pro Somatic instability at microsatellites, which consist of (CA),, repeats, vide signals for increased cell proliferation, which is suggested to be on chromosomes 5q, 15q, l'lp, and 18q occurs more frequently in necessary for the formation of aberrant crypts (16). It is possible that tumors of the proximal colon (44). Collectively, the human and suppressed mucosal immunity is also a component of the progression 5934

Antigen CryptEpithelium

Fig. 6. Proposed model for the mechanism of action of DMH-induced colon carcinogenesis in the suppression of immune-mediated ion transport in the distal colon. Colon carcinogenesis causes increased cr tissue expression of PGE@(47,48) and TGF-@3(49), both of which inhibit mediator release from mast cells (50, 51). As a result of carcinogenesis, PGE@is secreted from tissue macrophages (48), while TOF-@3isderived from both epithelial cells (49) and macrophages (54), as well as many other cell types. DMH-induced colon carcinogenesis also causes changes in the tissue levels of cAMP and protein kinase C (52, 53), second messengers important in mast cell function.

Table 1 Effect of DMH treatmentrejectionIntestinal on worm decreases tissue levels of cAMP (52) and protein kinase C (53) and increases levels of cGMP (52). Protein kinase C and cAMP are wormst'Groupâ€• important second messengers in mast cell function (54), and altered DMH3 Nonimmune Immune Immune + levels may lead to mast cell stabilization, constant degranulation and 80Â±9(15)'4 217Â±17(8) 75Â±11(8f mediator depletion, or down-regulation of the high affinity surface 14(2O)c5 208Â±21(10) 120Â±22(lOf 114Â± receptors for IgE. These possibilities, under current investigation in MÂ±11(12)'6 193Â±23(10) 69Â±10(11)' 101Â±14(9)'a 193Â±23(10) 69Â±10(11)' our laboratory, are summarized in Fig. 6. The mechanism by which larvaeafterGroupsofmicewereimmunizedbya primaryT.spiralisinfectionwith400 DMH changes levels of these important regulatory factors is not groups,DM11the second (group 3) or sixth (group 4) DMH injection. For the remaining known. DMH may act directly by inducing mutations of genes that beforethewas administered either 1 day before the primary infection (group 5) or 1day secondaryinfection (group6). control the synthesis of these factors. Alternatively, the carcinogen @ afterprimaryb number of worms establishing in the small intestine was determined 2 days may act indirectly by altering the production of other regulatory secondaryinfectioninfection with 400 T. spimlis larvae (nonimmune) or 2 days after molecules that in turn modulate the actions and synthesis of prostag asmean with 400 larvae (immune and immune + DMH). Worm counts are expressed Â±SE(n).C landins, TGF-f3, cAMP, CUMP, and protein kinase C. It is important group.ForSignificant difference (P < 0.05) compared with the respective nonimmune to note that prostaglandins (55) and TGF-f3 (54) are potent inhibitors theimmuneeach DM11 injection schedule, there was no significant difference between of many different immune reactions and that protein kinase C, cAMP, and immune + DMH groups. and cGMP are second messengers for many different cell types. Therefore, the results of such future studies should be relevant to other from normal colonic epithelium to the development of aberrantcrypts. aspects of mucosal immune responsiveness in addition to type I These results do not imply that type I hypersensitivity per se is hypersensitivity. important in the host defense against colonic neoplasia. Rather, this report demonstrates that early stages of colon carcinogenesis are REFERENCES capable of suppressing local immune function, which in the present I. Fearon, E. R., and Vogelstein, B. A genetic model for colorectal tumongenesis. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1994 American Association for Cancer Research. Early Stages of 1,2-Dimethylhydrazine-induced Colon Carcinogenesis Suppress Immune-regulated Ion Transport of Mouse Distal Colon

Russell R. Broaddus, Michael J. Wargovich and Gilbert A. Castro

Cancer Res 1994;54:5930-5936.

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