neuromedin N promoter

k-mediated activation neurotensin/neuromedin Nitesh A. Banker, MD, Mark R. Hellmich, and B. Mark Evers, MD, Galveston, Texas

of the human N promoter

PhD, Hong Jin Kim, MD, Courtney

M. Townsend,

Jr., MD,

Background. Expressionof the geneencodingthe neurotensin/neuromedinN (NT/N) is developmentally regulatedin the gut in a distinctive temporaland spatialfashion. Src kinase,a nonreceptortyrosinekinase, has beenimplicatedin thegrowth and differentiation of various tissues;its role in gut dafferentiation is not known. Thepurposeof this study was to determinewhetherthe 3-c signalingpathway plays a rolein the activation of the human NT/Npromoter Methods. Caco-2cells,a human colon cancercell line that can difjferentiateto a small bowelphenotype, weretransiently transfectedwith human NT/Npromoterfiagments linked to luciferuseand variousamountsof Src expression plasmidsor dominant negativeRaf; luciferuseand P-galactosidase activities weremeasuredafter 48 hours. Results.Cotransfectionof Src resultedin an approximateeightfoldincreaseof NT/N promoteractivity; mutation of a proximal activating protein-l/cyclic adenosinemonophosphate responsiveelementsite resultedin a dramatic decrease of Src-mediated NT/N induction. Cotransfectionwith a dominant negative Raf plasmidpartially blockedSrc-mediatedNT/N activation, Conclusions.Src increases NT/N promoteractivity in Caco-2cellsacting, in part, through a proximal AP-l/CRE promoterelement.In addition, Src regulation of the NT/N promoterappearsto bemediated through a Rafdependentpathway. Weproposethat Src mayplay a role in tissue-specajcic geneexpression in the gut. (Surgery 1997;122:180-6.) From

of Surgery,

the Department

The University

of Texas Medical

NEUROTENSIN, A TRIDECWEPTIDE RELEASED from spe-

cialized enteroendocrine cells (N cells) of the small bowel, has multiple functions in the gut including affecting secretion and motility and stimulating mucosal growth.‘” Expression of the gene encoding neurotensin and the structurally related hexapeptide neuromedin N (NT/N) is developmentally regulated in the guts of both rats and human beings in a distinctive temporal- and spatial-specific distribution.4,5 NT/N expression is initially low in the fetus but rapidly rises after birth to assume the distinctive adult distribution along the longitudinal axis of the small bowel. In the colon, NT/N is transiently expressed in the fetus when the colon is morphologically and functionalSupported (ROl

by grants from the National Institutes of Health DK48498, ROl AG10885, PO1 DK35608, T32 DK07639),

the UTMB Jeane B. Kempner Scholar Award Fund, and the James E. Thompson Memorial Foundation. Presented University

at the Fifty-eighth Surgeons, Tampa,

Annual Meeting of the Society Fla., Feb. 13-15, 1997.

of

Reprint requests: B. Mark Evers, MD, Department of Surgery, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0533. Copyright

0 1997 by Mosby-Year

0039-6060/97/$5.00

180

SURGERY

+0

Book,

11/6/81922

Inc.

Branch,

Galveston,

Texas

ly similar to the small bowel; however, expression of NT/N is not apparent in the colon of the newborn or the adult. To date, our findings have identified the NT/N gene as a potential molecular model to better delineate the mechanisms leading to differentiation of the normal small bowel and colon. The intracellular mechanisms regulating gut differentiation are complex and may depend on the interaction of underlying mesodermal components, specific transcription factors, and certain signal transduction pathways. Signaling mediators that may play prominent roles in gut differentiation include the Ras and Src families of proteins. Rasis a family of small guanine nucleotide-binding proteins localized to the inner surface of the plasma membrane; activated guanosine triphosphate-Ras associateswith c-Raf protein kinase to activate the mitogen-activated protein kinase cascade.6 Src kinasescomprise a family of nonreceptor kinasesthat transmit signalsby tyrosine phosphorylation through Rasdependent or ‘Ras-independent pathways7 Both Ras and Src have been implicated in the growth and differentiation of various tissues. For example, in the normal bowel Rasis expressed in an increasing gradient along the normal gut

181

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Banker et al.

-373

NT/N-Luc pMvSrc

(Fg) (pg)

5.0 -

1.0

2.5

5.0

-

-

b -

PMcSrc

(pg)

-

-

-

-

1.0

2.5

5.0

Fig. 1. Human NT/N promoter (-373/+26)-luciferase plasmid (5 bg) was transiently 2 cells with varying amounts of either activated viral Src (pMvSrc; single-hatched (PM&-c; closed bars). Amount of DNA was kept constant by adding empty vector expressed as the fold mean * standard deviation (SD) of the fold induction of NT/N fections.

NT/N Promoter

Fold NT/N Promoter

Deletions

I

0

2

4

6

6

transfected into Cacobars) or normal C-SK DNA (pEVX). Data are for four separate trans-

Activation 10

12

-373 -m

-373

171

-m

-122x

LUC

-424

QO--jg

I

0 m

PE= pMvSrc

Fig. 2. Human NT/N promoter-luciferase 5’ deletions tr-ansiently transfected into Caco-2 cells with either the empty vector (pEVX; open bars) or the Src expression plasmid (pMvSrc; closed bars). Data are expressed as the fold mean f SD of fold induction for four separate transfection experiments.

axis, with the highest levels in the more differentiated cell types such as the villus tip cells and endocrine cells.* In addition, stable transfection of activated Ha-ras induces a morphologic differentiation of a human colon cancer line, Caco-2, to a small bowel phenotype. g Furthermore, we have recently noted that the Ras/Raf signaling pathway may play a role in NT/N gene activation as demonstrated by induction of NT/N gene expression in Caco-2 cells stably transfected with Ras.‘O The Src kinases have been linked to differentiation of neurons, keratinocytes, and hemopoietic cells11,12; however, the role of Src in normal gut development is not known.

The purpose of our study was to determine whether Src kinase plays a role in the activation of the human NT/N promoter and whether this activation is dependent on the Ras/Raf signaling pathway.

MATERIAL AND METHODS Material. Restriction and other

DNA-modifying enzymes were purchased from Promega Corp. (Madison, Wis.) or Gibco-BRL (Grand Island, N.Y.) . All plasmids were prepared with a Quiagen plasmid purification kit (Quiagen, Inc., Chatsworthy, Calif.). All other reagents were of molecular biology grade and were obtained from either Sigma Chemical CO.

Surgery

Banker et al. 182

Volume 122, Number2

NT/N Promoter

Mutation

Fold NT/N Promoter 1 q 3 P

Activation 4 6 6

0 m

PEVX pMvSrc

fig. 3. Wild-type human NT/N promoter fragment (-175/+4) and a 10 bp linker scanning mutation of a proximal AF’-l/CRE domain (-50/-41) were transiently transfected into Caco-2 cells with either empty vector (pEVX; open bars) or the Src expression plasmid (pMvSrc; closed bars). Data are expressed as the mean fold + SD of the fold for five separate transfections.

-373 NT/N-Luc

(pg)

PMvSrc (kg) Raf-C4 (pg)

5.0 -

b 2.5 -+ -

1.25

2.5

Fig. 4. Human NT/N promoter (-373/+26)-luciferase plasmid was transiently transfected into Caco-2 cells with varying amounts of Raf-C4 (Raf dominant negative; single-hatched bars) along with either empty vector (pEVX) or Src (pMvSrc). Amount of DNA was kept constant by adding the empty vectors pEVX and pkRSA. Data are expressed as the mean fold f SD of the fold for two transfection experiments performed in duplicate.

(St. Louis, MO.) or Fisher Biotechnology (Fair Lawn, N.J.) . Tissue culture media, fetal calf serum, and supplements were obtained from Gibco or Hyclone Laboratories (Logan, Utah). ‘Gsue

culture.

The human

colon

cancer

(Cacclt2)

cell line wasobtained from the American Type Culture Collection (Rockville, Md.) and maintained in minimal essential medium enriched with nonessential amino acids and sodium pyruvate and supplemented with 15% (vol/vol) fetal calf serum in a humidified atmosphere of 95% air and 5% CO, at 37” C. Transient transfection, luciferase, and j%galactosidase assays. The human NT/N promoter frag-

ment (-373/+26) subcloned into pXP1 luciferase vector came from Dr. Paul Dobner (University of Massachusetts,Worcester, Mass.). Human NT/N 5’ deletions

and linker

scanning

mutations

were

con-

structed by methods similar to those reported previously’3 and will be described in detail elsewhere (unpublished data). Src kinase constructs, normal c-Src (pMcSrc) , and the activated viral Src (pMvSrc) came from Dr. David Shalloway (Cornell University, Ithica, N.Y.). l4 The dominant negative Raf construct, Raf-C4, was obtained from Dr. Ulf Rapp (National Cancer Institute, Frederick, Md .) .l5 For each transfection experiment, efflcien-

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SUP? August 1997

cy of transfection was assessedby cotransfection with a plasmid (CMV-j3-gal) encoding P-galactosidase. For the transient transfection assays,Caco-2 cells (passages20 to 30) were plated in 60 mm dishes 36 hours before transfection. Human NT/N promoter-luciferase gene constructs (5 kg) were cotransfected with varying amounts of Src expression plasmids or the Raf-dominant negative plasmid by calcium phosphate coprecipitation as we have described previously. l”,13 The amount of DNA transfected waskept constant by adding empty vector as needed. Forty-eight hours after transfection, cells were lysed and assayed for luciferase with a Monolight 2010 (Analytical Luminescence Laboratory, San Diego, Calif.) luminometer. Both the luciferase and P-galactosidaseassayswere performed as described previously.‘” RESULTS Src kinase activates the human NT/N promoter, in part, through a proximal activating protein1 /cyclic adenosine monophosphate responsive element (AI’-l/CRE) domain. To determine whether

Src kinase activates the human NT/N promoter, Caco-2 cells, human colon cancer cell lines that can differentiate to small bowel phenotype, were transiently transfected with the human promoter fragment (-373/+26) linked to a luciferase reporter gene along with increasing amounts of Src expression vector (Fig. 1). A fourfold to eightfold activation of the NT/N promoter was noted with the addition of viral Src (pMvSrc). By comparison, a twofold to threefold activation was noted with normal c-Src (pMcSrc). Taken together, these findings provide support for a role for Src in the regulation of the NT/N gene. To delineate the regions of the NT/N promoter that are important to Src-mediated activation, transient transfection assayswith a series of 5’ deletion sequencesof the human NT/N promoter linked to luciferase were performed (Fig. 2). The proximal 171 basepair (bp) of the 5’ flanking region wassufficient to obtain maximal reporter gene expression. Deletion of regions between -373 and -171 doubled NT/N reporter activity compared with the -373 promoter fragment, suggesting the presence of a repressor element in this region. Further deletion of regions between -171 and -122 returned NT/N reporter activity to levels similar to the -373 construct. Deletion of basesbetween -122 and -42, which includes a proximal Al’-l/CRE site located at -48 to -42 bp from the transcriptional start site, resulted in the loss of Src-mediated activation of the NT/N promoter. These results suggest that the

AP-l/CRE domain is important in Src-mediated NT/N activation. To better ascertain whether this proximal AP1/CRE domain is required for Src-mediated activation, a 10 bp linker scanning mutation that alters this domain was constructed and linked to the luciferase reporter gene. Assays were performed with either this mutated NT/N construct (-50/-41 NT/N-luciferase) or the wild-type NT/N promoter fragment (-175/+4) cotransfected with the Src expression plasmid (Fig. 3). Compared with the wild-type NT/N, mutation of the proximal APl/CRE domain significantly diminished Src-mediated NT/N reporter activation. Collectively, these data demonstrate that Src-mediated activation is, in part, regulated by the proximal AP-l/CRE domain. Src-mediated tional Ras/Raf

NT/N activation requires a funcpathway. Previously, we have

demonstrated that overexpression of Ha-rus increased NT/N promoter activation and that this induction was blocked by cotransfection with dominant negative Raf. lo These findings have placed Raf downstream from Rasin the pathway leading to NT/N expression. To determine whether Src kinase mediates NT/N promoter activation through a Ras/Raf-dependent pathway, transient transfection assayswere performed with the -373 NT/N promoter fragment and the Src expression plasmid along with varying amounts of a Raf construct encoding a dominant negative Raf (Raf-C4; Fig. 4). Cotransfection of Raf-C4 inhibited Srcmediated activation of NT/N in a dose-dependent fashion. These findings suggest that the Ras/Raf pathway lies downstream of Src in the signaling pathway that leads to NT/N induction. DISCUSSION

The complex process of gut differentiation remains poorly understood largely because of a multilevel interaction of numerous signal transduction pathways and activation of asyet undefined tissue-specific genes. In this study we found that the terminally differentiated gut endocrine gene, NT/N, is activated by overexpression of Src. Furthermore, this Src-mediated NT/N activation appears mediated, in part, by a R&dependent pathway with signalsconverging on a crucial AP-l/CRE promoter element. Consistent with our results, other investigators have shown that Src can affect expression of various downstream target genes through different c&acting elements including AP-1, l6 the serum-responsiveelement,” and the activating transcription factor/CRE domain.” The diversity of the c®ulatory domains regulated by Src underscores the possibility that tis-

Banker et al. 184

SUWY Volume 122, Number 2 sue-specific factors and environment are likely contributors to the distinct regulation of target genes. Because none of the previous studies examined tissue-specific genes, our findings provide the only evidence for a role for Src in gut-specific gene expression. To delineate whether Src is activating the NT/N promoter through the Ras/Raf pathway or, alternatively, through an independent pathway, we used a dominant negative Rafplasmid and found a dosedependent inhibition of Src-mediated NT/N activation. In this study we infer the role of Ras in mediating Src signal to Raf indirectly; asother studies have shown, inhibition of Src mediated promoter activity of other genes by blocking R~.s.‘~,I9 Our findings of an apparent Src/Ras/Raf signaling cascade leading ultimately to gene induction are also consistent with the findings of others in models of differentiation (e.g., nerve growth factor-induced PC12 cell neurite outgrowth) and in the signal transduction of mitogenic stimuli.lgM2’ Our findings suggest that Src is acting, in large part, through an intact Ras/Raf signaling pathway to activate NT/N; however, we cannot entirely exclude the possibility that Src may induce NT/N activity through additional pathways that are independent of Ras/Raf. Alternatively, it is plausible that Src may directly activate Raf, but current evidence does not support Raf activation independent of bs

6,15,19,20

Previous studies have established a role for Src in colon carcinogenesis22,23; the progressive increase in Src protein levels and activity correlates with the development of premalignant statesto carcinoma. However, the role of Src in normal gut development is not defined. Burgess et a1.24 observed that crypt cells from the adult chicken duodenum contain higher levels of tyrosine phosphorylated proteins than do differentiated enterocytes at the villus tips; however, they did not directly link Src activity to the tyrosine phosphorylated protein pools. The only direct evidence implicating Src activity in the gut is provided by Cartwright et a1.,25 who observed that undifferentiated crypt cells contained significantly higher levels of cytoskeletal-associated Src protein tyrosine kinase activity than differentiated cells of the gut. Collectively, these observations suggest that the maturation of enterocyte from the crypt to the villus is associatedwith a decline in Src kinase activity. In contrast, we have shown that Src can activate NT/N, a localized and terminally differentiated endocrine gene of the gut. These findings may represent a dichotomy in the cellular function of the Src signaling pathway in the normal mucosal

growth from differentiation of the gut. Future studies are required to better elucidate the exact role of normal Src in the differentiation process and in the ultimate expression pattern of other differentiated gut. genes. In conclusion, our study suggestsa role for the Src pathway in regulating the gut-specific NT/N gene and further underscores the importance of the Src/Ras/Raf pathway in this process. We propose that Src may play a role in tissue-specific gene expression in the gut and, possibly, in the process of gut differentiation. REFERENCES 1. Ferris CF, Carraway RE, Hammer RA, Leeman SE. Release and degradation of neurotensin during perfusion of rat small intestine with lipid. Regul Pept 1985;12:101-11. 2. Feurle GE, Mtiller B, Rix E. Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats. Gut 1987;28(1 suppl):19-23. 3. Wood JG, Hoang HD, Bussjaeger LJ, Solomon TE. Neurotensin stimulates growth of small intestine in rats. Am J Physiol 1988;255:G8137. 4. Evers BM, Ehrenfried JA, Wang X, Townsend CM Jr, Thompson JC. Temporal-specific and spatial-specific patterns of neurotensin gene expression in the small bowel. Am J Physiol 1994;267:G875-82. 5. Evers BM, Rajaraman S, Chung DH, Townsend CM Jr, Wang X, Graves K, et al. Differential expression of the neurotensin gene in the developing rat and human gastrointestinal tract. Am J Physiol 1993;265:G482-90. 6. Koide H, Satoh T, Nakafuku M, Kaziro Y. GTPdependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc Nat1 Acad Sci USA 1993;90:86836. 7. Erpel T, Courtneidge SA. Src family protein tyrosine kinases and cellular signal transduction pathways. Curr Opin Cell Biol 1995;7:17682. 8. Furth ME, Aldrich TH, Cordon-Cardo C. Expression of ras proto-oncogene proteins in normal human tissues. Oncogene 1987;1:47-58. 9. Celano P, Berchtold CM, Mabry M, Carroll M, Sidransky D, Casero RA Jr, et al. Induction of markers of normal differentiation in human colon carcinoma cells by the v-mp oncogene. Cell Growth Differ 1993;4:341-7. 10. Evers BM, Zhou Z, Celano P, Li J. The neurotensin gene is a downstream target for Ras activation. J Clin Invest 1995;95:2822-30. 11. Kefalas P, Brown TRP, Brickell PM. Signalling by the ~60’~‘” family of protein-tyrosine kinases. Int J Biorhem Cell Biol 1995;27:551-63. 12. Zhao Y, Sudol M, Hanafusa H, Krueger J. Increased tyrosine kinase activity of c-.%-c during calcium-induced keratinocyte differentiation. Proc Nat1 Acad Sci USA 1992;89:8298-302. 13. Evers BM, Wang X, Zhao Z, Townsend CM Jr, McNeil GP, Dobner PR. Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line. Mol Cell Biol 1995;15:3870-81, 14. Johnson PJ, Coussens PM, Danko AV, Shalloway D. Overexpressed pp60c”‘c can induce focus formation with-

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15.

16.

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18.

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20.

21.

22.

23.

24.

25.

out complete transformation of NIH 3T3 cells. Mol Cell Biol 1985;5:1073-83. Bruder JT, IIeidecker G, Rapp UR. Serum-, TPA-, and Rasinduced expression from Ap-l/Et+driven promoters requires Raf-1 kinase. Genes Dev 1992;6:545-56. .%tO H, Kita M, Seiki M. v&-c activates the expression of 9% kDa Type IV collagenase gene through the AF-I site and the GT box homologous to retinoblastoma control elements. J Biol Chem 1993;268:23460-8. Qureshi SA, Cao X, Sukhatme VP, Foster DA. v-Src activates mitogen-responsive transcription factor Egr-1 via serum response elements. J Biol Chem 1991;266:10802-6. Xie W, Fletcher BS, Andersen RD, Herschman HR. v-STC induction of the TISIO/PGSS prostaglandin synthase gene is mediated by an ATF/CRE transcription response element. Mol Cell Biol 1994;14:6531-9. Pickrtt CA, GurierreT-Hartmann A. Ras mediates Src but not epidermal growth factor-receptor tyrosine kinase signaling pathways in GH4 neuroendocrine cells. Proc Nat1 Acad Sci USA 1994;91:8612-6. Kolch W, Heidecker G, Lloyd P, Rapp UR. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 1991;349:426-8. D’Arcangelo G, Halegoua S. A branched signaling pathway for nerve growth factor is revealed by Src-, Ras, and Rafmediated gene inductions. Mol Cell Biol 1993;13:314&5.5. Bolen JB, Veillette A, Schwartz AM, DeSeau V, Rosen N. Activation of pp60c-“” protein kinase activity in human colon carcinoma. Proc Nat1 Acad Sci USA 1987;84:2251-5. Cartwright CA, Meisler AI, Eckhart W. Activation of the p~60’+~’ protein kinase is an early event in colonic carcinogenesis. Proc Nat1 Acad Sci USA 1990;87:558-62. Burgess DR, Jiang WP, Mamajiwalla S, Kinsey W. Intestinal crypt stem cells possess high levels of cytoskeleta-associated phosphotyrosine-containing proteins and tyrosine kinase activity relative to differentiated enterocytes. J Cell Biol 1989;109:2139-44. Cartwright CA, Mamajiwalla S, Skolnick SA, Eckhart W, Burgess DR. Intestinal crypt cells contain higher levels of rytoskeletal-associated pp60c-“” protein tyrosine kinase activity than do differentiated enterocytes. Oncogene 1993;8:1033-9.

DISCUSSION Dr. Richard A. Hodin (Boston, Mass.) Your laboratory has really done a great job in putting the neurotensin gene on the map and showing that it is an excellent tool to use in unraveling the fundamental mechanisms of gut growth and differentiation. First, my understanding is that in previous work you have been ahle to show that stably transfected cells expressing the ras gene were able to induce the endogenous neurotensin gene. Have you had a chance to do the same kind of studies with the src gene? Does it really turn on the endogenous neurotensin gene or are your results more a function of the transfection system? Second, in the case of both the src and your ras experiments, are the effects seen in nonintestinal cells? Have you had a chance to look at cell lines other than Caco-2 cells? Dr. Banker. In answer to your first question, we are currently working on stably transfecting srcinto the Caco2 cell line to determine exactly the same question you asked, and whether there can be constitutive expres-

sion with stable transfection of SK In answer to your second question, work done by Dr. Evers in our laboratory has also focused on a pancreatic endocrine cell line called BON that constitutively expresses the neurotensin gene. We have not specifically looked for neurotensin expression in non-gut-related cell lines. Dr. Barbara L. Bass (Baltimore, Md.). Obviously, the big question that you are trying to answer is what regulates gut differentiation. To that end you have chosen the neurotensin gene as a marker of differentiation, and you have used the Caco-2 cell line, which is a good one because it will differentiate. In your studies in which you have altered signaling pathways, inducing the Src pathway, for example, do you induce markers of differentiation? Have you looked for or found, for example, alterations in disaccharidase activity or other markers like alkaline phosphatase or villin, which are more generally accepted markers of differentiation? In addition, when you alter this signaling pathway, do you see morphologic changes in enterocyte phenotype, such as polarity, that are consistent with the differentiated or undifferentiated enterocyte? Last, I would like you to take a mechanistic step backward from this intracellular signaling pathway and speculate for us about the intestinal milieu and how in fact we get this process started. What is it in the crypt villus axis that allows initiation of the Src signaling pathway to ultimately lead to this altered regulation of differentiated gene expression? Dr. Banker. Those are quite important questions. We have not as yet examined the other intestinal-related genes or resulting changes in phenotype with regard to STCexpression. Dr. Evers has already looked at sucrase-isomaltase in regard to rus stably transfected Caco-2 cell lines. But those experiments you suggest will need to be done in the future with alterations of Src in the pathway. With regard to your second question about mechanistic approach, we speculate that the underlying mesenchyma1 factors, along with specific transcription factors that would be activated, may lead to specific differentiation into small bowel versus the colonic phenotypes. Dr. De&n Fleming (Houston, Texas). In our laboratories we have been studying the functions of the Src family members, the protein tyrosine kinases, in the progression of colorectal carcinoma, and I would like to ask a couple of questions in that regard. First, we have seen that other Src family members can be elevated in their kinase activity in human colorectal cancers, specifically cYes. Have you or anyone in your laboratory done any studies to examine whether there is a potential role of other Src family members in the regulation of the neurotcnsin gene? Second, I know that a previous study by Dr. Evers specifically examined the ontogeny of neurotensin expression during fetal gut development. Are there are any studies that you have done or that are ongoing that are looking at the expression of SK? Finally, Src family members are expressed at an abnormally high amount in approximately 90% of human colorectal cancers, Src, Yes, or a combination of them. A previous study from your laboratory looking at neu-

Banker et al. 186

SufP-J Volume 122, Number 2 rotensin expression demonstrated that it was up-regulated in about 25%. I know that it is a complex matter to figure out what is going into this, but could you speculate on some of the other factors that might be contributing to the expression of neurotensin in colorectal malignancies? Dr. Banker. In response to your first question, we are beginning to explore other members of the Src family, particularly c-Yes, to examine whether there is any overlap in neurotensin gene regulation between the family members. We do not have any data to show you yet. In regard to your second question, the ontogeny of Src in the gut has not been previously examined. We have begun to do so and have preliminary results indicating that STCmessage is present in the developing gut

and in the adults. In regard to your third question, you remind us that only 25% of the colon cancers have been found with neurotensin expression, whereas up to 80% of colon cancers have elevated Src family members. The disparity has to be explained by one of several possibilities. Obviously, src expression alone is not the answer, nor is elevation of rus as found in some of the colon cancers. It must involve multifactorial interaction of signal transduction pathways with underlying specific transcription factors, thereby activating tissue-specific genes leading to expression of neurotensin. The basic answer may be that colon cancers exhibit various levels of differentiation and may not have all the underlying factors necessary to express the neurotensin gene.

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