Characterization of preneoplastic and neoplastic lesions in the rat ...

American Journal of Pathology, Vol. 138, No. 2, February 1991 Co>pight X Ametican Association of Pathologists

Characterization of Preneoplastic and Neoplastic Lesions in the Rat Pancreas Daniel S. Longnecker,* Olive S. Pettengill,* Bruce H. Davis,* Barbara K. Schaeffer,* Joanne Zurlo,t H. Lily Hong,4 and Elna T. Kuhlmann* From the Departments of Pathology* and Pharmacology and Toxicologyt, Dartmouth Medical School, Hanover, New Hampshire; and the Chemical Pathology Branchb, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Nodules of acinar cells with increased proliferative potential develop in the pancreases of carcinogentreated rats and in untreated aged rats Large nodules are classed as adenomas. Phenotypic and genotypic characteristics of nodule cells were compared with normal pancreas and transplantable acinar cell carcinomas by several methods. Nuclei of acinar cells from normal pancreas, adenomas, and three carcinomas in situ had normal diploid DNA content as determined by flow cytometry. One of two primary carcinomas had a hypodiploid DNA content. Two of three transplantable carcinomas were aneuploid with a DNA content in the tetraploid range. Explants from nodules and adenomas failed to grow in soft agar, whereas several carcinomas were positive in this assay. A primary carcinoma was serially transplanted4 but transplantation of nodules or adenomas failed Transfection of DNA from carcinomas in situ yielded a higher frequency of NIH 3T3 transformants than DNA from adenomas. DNAs from the transformants did not contain ras sequences These studies indicate that cells from nodules and adenomas have low growth potential and lack criticalphenotypic and genotypic charactertistics of transformed malignant cells that were present in some primary and transplanted carcinomas. (Am J Pathol 1991,

138:333-340)

Several animal models for study of pancreatic carcinogenesis have been developed in recent years.1 These provide evidence that carcinogenesis in the pancreas, as in other organs, proceeds by the clonal expansion of sin-

gle initiated cells through a sequence of subsequent steps that give rise to fully developed carcinomas.1'2 Focal proliferative lesions develop in the pancreases of carcinogen-treated rats3 and in untreated, aged ratsespecially those fed high levels of corn oil.4 These lesions have been especially well characterized in the azaserineinduced rat model of pancreatic carcinogenesis.2 Some clones appear to have greater growth potential from the time of initiation or shortly thereafter so that they grow to become large nodules of apparently homogeneous phenotype, the largest of which are classed as adenomas.2 Other clones (nodules) appear to undergo a stepwise progression that can be detected as a series of secondary or tertiary phenotypic changes that can be detected morphologically by altered (usually diminished) differentiation.3 We have referred to the earliest grossly identifiable lesion in the carcinogen-treated pancreas as an atypical acinar cell nodule.2 The practical lower size limit for gross identification of such nodules is about 1 mm. These are composed of atypical, but well-differentiated, acinar cells. Smaller lesions, detected only microscopically, are designated as foci. Those nodules that are 3 mm or greater in diameter are designated as adenomas, ie, lesions usually in the size range of 3 to 6 mm in diameter that are composed of a monomorphic population of welldifferentiated acinar cells. Microscopic foci or nodules measuring 0.5 mm or more in diameter that are apparently localized tumors with anaplastic cellular changes are classed as carcinoma in situ (CIS) when the degree of anaplasia clearly suggests malignant change and especially when there is evidence of desmoplasia within or at the margin of the lesion. The classification criteria for azaserine-induced lesions have been described and the lesions illustrated in several earlier reports.2 Neoplasms with evidence of local invasion or metastasis are designated as carcinomas. In the studies reported here, lesions representing varSupported by NIH grants ES-03687, ES-03283, and CA 23108, USPHS. This work was part of a Cooperative Agreement supported by NIEHS for which Dr. Gary A. Boornan served as project officer. Accepted for publication September 14, 1990. Address reprint requests to D. S. Longnecker, Department of Pathology, Dartmouth Medical School, Hanover, NH 03756.

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ious stages of neoplastic progression were characterized by several approaches in an attempt to identify subpopulations with high potential for growth and progression.

Materials and Methods The approaches used to characterize nodule phenotype and genotype included histology, determination of nuclear DNA content by flow cytometry, cell culture (growth in soft agar), transfection of DNA into NIH 3T3 cells, and transplantation (tumorigenicity). Tissue samples were obtained from rats that were killed by decapitation after ether anesthesia. Necropsies were done promptly. Using carcinogen-treated animals as donors, a spectrum of lesions found at various times after treatment were collected. Similar lesions from non-carcinogen-treated, corn-oi[-gavaged rats were evaluated for tumorigenicity. A series of transplantable azaserine-induced acinar cell carcinomas were included as 'positive' controls.

Normal Pancreas Individual samples of normal pancreas were removed from 12- to 1 5-week-old male Lewis (LEW) rats.

Nodules Male LEW and F344 rats were obtained from the Charles River Breeding Laboratories, Wilmington, Massachusetts, as nursing pups. They were injected intraperitoneally at age 14 (LEW) or 14 and 21 (F344) days with 30 mg azaserine/kg body weight (Calbiochem-Behring Corp, La Jolla, CA) or N-nitroso(2-hydroxypropyl)(2oxopropyl)amine (HPOP), 80 mg/kg (LEW), or 160 mg/kg (F344), dissolved in 0.9% saline at age 14 days. The HPOP was a gift of Dr. J. E. Saavedra, Frederick Cancer Research Center, Frederick, Maryland. The rats were weaned to AIN-76A diet (Teklad, Madison, WI) containing 5% (LEW) or 20% (F344) corn oil. These 'donor' rats were killed 12 to 18 months after injection with azaserine or HPOP and the nodules were dissected free from the surrounding normal pancreas tissue. Each nodule was weighed, and, in most cases, a representative portion was removed for histology. The remainder was frozen for flow cytometry or used for other studies. Nodules of similar size were pooled, when necessary, to obtain amounts of tissue larger than could be provided by single nodules. In a separate set of studies, male F344 rats at 5 to 7 weeks of age were gavaged with corn oil, 10 ml/kg body weight, 5 days/week for 85 to 112 weeks to induce nodules and adenomas for study.

Carcinomas Transplantable rat pancreatic tumor tissue from three different carcinomas maintained in this laboratory by serial transplantation with intermittent cryopreservation2'5'6 were harvested for analysis. The transplantable carcinomas all originated in the pancreas of azaserine-treated rats.

Samples for flow cytometry were frozen in 40 mmol/l (millimolar) citrate buffer, pH 7.6, with 250 mmol/l sucrose containing 5% dimethyl sulfoxide (DMSO)7 and stored in a - 70°C freezer until nuclei were prepared. Other samples of tissue were frozen for isolation of DNA, or fresh tissue (removed aseptically) was placed in culture or used for transplantation. Tissue for histology was fixed in Bouin's solution, embedded in paraffin, and stained with hematoxylin and eosin (H&E).

Preparation of Nuclei for Flow Cytometry Tissues were quickly thawed at 370C, minced in phosphate-buffered saline ethylenediaminetetra-acetic acid (PBS-EDTA) buffer, and homogenized in a Dounce homogenizer. The homogenate was filtered through 64and 25-,u Nytex mesh and centrifuged. The resulting nuclear pellet was resuspended in PBS with 20 mmol/l MgCI2 and held at room temperature for 30 minutes to allow RNA digestion by endogenous ribonuclease. Preparation of tumor samples included addition of 1 mg/ml RNAse A, type 1 A (Sigma Chemical Co., St. Louis, MO) during the room temperature incubation because of the low endogenous RNAse content in the tumors. The suspension was then centrifuged and the supernate replaced with sucrose-citrate buffer to a nuclear concentration of 5 x 1 05 per milliliter. The nuclei were stained with propidium iodide in the three-step method of Vindelov et al.7 All samples were prepared in duplicate, with one of the pair containing an internal DNA standard of chick red blood cells (CRBC). DNA content was determined by flow cytometry using a Coulter EPICS 541 flow cytometer. Twenty thousand nuclei were analyzed in the samples containing the CRBC and 50,000 in those without. The coefficients of variation of the G1/GQ peaks ranged from 1.95 to 3.84. An index of pancreatic cell DNA content was calculated using CRBC as a standard and setting the mean of 10 normal pancreas samples as 1 (the standard deviation [SD] was 0.01). An index greater or less than 1 ± 2 SD was taken to indicate a hyperdiploid or hypodiploid DNA content.

Growth in Soft Agar Individual carcinomas, large individual adenomas, and pooled smaller adenomas or nodules were minced and

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then placed in growth medium. Eight pools were used because the sample size required for effective processing of specimens for the soft agar assay required more tissue than could be provided by single lesions. The optimal medium was composed of Hams F12 (Gibco, Grand Island, NY), NuSerum (Collaborative Research, Lexington, MA), carbachol, and secretin (Sigma), soybean trypsin inhibitor (Cooper Biomedical, Malvern, PA), caerulein (a gift of Dr. R. deCastiglione, Farmitalia, Milan, Italy), and 0.36% SeaKem Agarose (Marine Colloids, Rockland, ME). Plates were observed for evidence of growth for 3 weeks and colonies showing cell growth by phase microscopic observation were rated as positive. Selected colonies were picked and fixed for light and electron microscopic examination and were found to exhibit acinar cell morphology.

Transplantation Studies Individual lesions were dissected from the pancreas and a portion was taken for histologic study. The major portion was minced in Hanks balanced salt solution or tissue culture medium, and 0.3 ml of the suspension containing several tissue fragments was injected subcutaneously or intraperitoneally into 3- to 6-week-old syngeneic male rats. For renal subcapsular implants, a single 1-mm fragment was inserted beneath the renal capsule through a flank incision using a trochar. Recipients were palpated weekly for evidence of SC tumor growth and killed when clearly positive or after 6 months if negative. Growth of transplanted tissue was confirmed histologically. In the study with corn-oi[-gavaged rats, necropsies were done on six rats after 85 weeks of treatment, and the two largest pancreatic nodules seen grossly from each donor were placed in a sterile petri dish in saline. A 1 -mm cube of tissue from each nodule was transplanted under the kidney capsule of four recipients with the remainder of the nodule fixed in 10% buffered formalin for histologic examination. The recipients were 4- to 5-week-old male F344 rats that had been anesthetized with a combination of ketamine (100 mg/ml) and xylazine (3.2 mg/ml) intramuscularly at 0.1 ml per 100 g body weight. The hair was shaved, a midflank incision made, and the left kidney exposed. A nick was made in the kidney capsule at the caudal pole and the tumor transplant placed under the kidney capsule toward the anterior pole. The abdominal wall was closed with continuous sutures of catgut and the skin closed with stainless steel wound clips. Three months later, necropsies were done on the recipients and the kidneys examined. Any grossly visible nodule was retransplanted. This procedure was repeated for six male F344 rats that had been gavaged with corn oil for 112 weeks.

DNA Transfection Studies DNA was isolated from three adenomas and two CIS and transfected into NIH 3T3 cells.

DNA Preparation DNA was prepared by sodium dodecyl sulfate/ proteinase K lysis and phenolchloroform extraction.8

Transfection NIH 3T3 cells were cotransfected with 20- to 30-p.g nodule DNA along with 75 ng of the plasmid, pSV2-neo (American Type Culture Collection, Rockville, MD) as a calcium phosphate precipitate.9 10 Transformants were selected by growth in medium containing the antibiotic G-418 (Gibco) as previously described.10

Southern Blot Hybridization DNA samples were digested with restriction endonucleases, subjected to agarose gel electrophoresis and transferred onto Gene Screen Plus nylon membranes (NEN Research Products, Boston, MA) by the method of Chomzynski and Qasba.11 DNA blots were hybridized with rat repetitive sequence 3B5, v-K-ras (Hi Hi 380) and v-H-ras (BS9), and N-ras (exons and 11) that were 3p labeled by oligonucleotide elongation.12 K-ras and H-ras were gifts of Dr. R. Ellis (Merck, Sharp and Dohme, West Point, PA). N-ras was obtained from ATCC. 3B5 was a gift from Dr. A. Furano (NIH, Bethesda, MD). Hybridization was carried out at high stringency in 50% formamide.

Identification of Transforming Genes Southern blots of restriction endonuclease digests of transformant DNAs from eight foci were probed with 3B5, the probe for rat repetitive DNA and with pSV2neo. Xbal and Bam HI digests were probed with v-H-ras. Xbal digests were probed with v-K-ras and N-ras.

Results Lesions were classified on the basis of size, gross characteristics, and histologic criteria as indicated above and reported previously.1' 2 The lesions were classified histologically when size permitted. Those lesions used for flow cytometry without sampling for histology were all in the

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adenoma category on the basis of their size, but are listed separately in the tables. A relative DNA content of 2.44 ± 0.02 for pancreatic nuclei was calculated using the ratio between the G1/Go peak from each of the 10 normal pancreases and the CRBC peak. When the control nuclear DNA contents were normalized to the mean value, the standard deviation was 0.01. A DNA index normalized to the mean value was calculated for each nodule or tumor sample. An index of greater than 1.02 was classified as an increased or hyperdiploid amount of DNA, and an index of less than 0.98 was taken to indicate a hypodiploid DNA content. Of the lesions classified as adenomas, by either histology or size, or as CIS, all fell within two standard deviations of the 2N DNA content defined for normal acinar cells (Table 1). One of two primary carcinomas was hypodiploid (Figure 1). Two of three transplantable acinar cell carcinomas had DNA content in the tetraploid range. These results are reported in Table 1. Explants from nodules failed to grow in soft agar, whereas several carcinomas were positive in this assay (Table 2). Six attempts to grow nodules and adenomas in monolayer culture were unsuccessful, as was one attempt to grow a carcinoma in situ. One cell line was established from a well-differentiated, azaserine-induced acinar cell carcinoma that was serially transplanted as part of this experiment. This is the sixth azaserineinduced carcinoma that has been successfully transplanted in our laboratory. There was no growth after transplantation of carcinogen-induced nodules or adenomas. Transplantation of CIS or carcinomas gave a low yield of growth in the recipients (Table 3). A total of 12 nodules from rats given Table 1. Nuclear DNA Content in Nodules and Carcinomas Weight

Specimen Adenomas By histology (n = 11) By size (n = 8) Carcinoma in situ

Primary carcinoma Transplanted carcinoma no.§ DSL-3 DSL-4 DSL-6

(mg)t

corn oil for 85 weeks and 12 nodules from rats given corn oil for 112 weeks were transplanted into 96 recipients. Histologically, 8 of the 12 nodules transplanted at 85 weeks were confirmed to be adenomas, two were focal hyperplasias, and in two cases not enough material remained to be diagnostic, which suggests that they were small lesions, ie, nodules. At 1 12 weeks, 9 of the 12 nodules were confirmed to be acinar cell adenomas, 1 was an islet cell adenoma, and 2 were normal pancreatic tissue, ie, not tumors. Thus a total of 17 acinar cell adenomas were transplanted to 68 recipients. Most were acinar cell adenomas composed of well-differentiated cells (Figure 2). Histologically the adenomas showed increased mitotic activity, moderate atypia, and an increased number of pyknotic nuclei. When the necropsies were done on the recipients after 3 months, no tissue remained in most recipients. Histologically only a small increase in fibrous tissue and inflammatory cells were found at the transplant site. In three recipients, transplanted tissue remained but did not appear to have grown. This residual tissue was transplanted (each to two recipients). Histologically the transplant appeared more differentiated than the original nodule. No growth was seen in the second set of recipients when necropsies were done 3 months later. Transfection of DNA from adenomas yielded a low frequency of transformed colonies (within background), whereas DNA from CIS yielded a higher frequency of transformants (Table 4). Southern blots of DNA digests from the eight transformed foci were positive when probed for rat repetitive DNA and pSV2neo. DNAs from the transformants were negative when screened for rat H-, N-, and c-K-ras. None of the ras probes hybridized to bands other than to those corresponding to the endogenous mouse genes in the NIH 3T3 cells (results not shown).

DNA index

Discussion 25-132 33-65 73 85 130 66 1000

1.00 ± 0.01t 1.00 ± 0.0t 1.00 0.99 0.99

0.81t 1.00 1.89 ± 0.00t 2.28 ± 0.03t 1.01 ± 0.01

* The range is defined for adenomas, and weights for individual lesions are given for CIS and primary carcinomas. t The mean DNA content of nuclei from normal pancreas is defined as 1, as discussed in the text. The mean index for 10 normal pancreases was 1.00 ± 0.01 (SD). t Aneuploid DNA content, as defined in the text. § DSL-3 and DSL-4 were derived years ago and have been transplanted many times. DSL-6 was established during this study and was studied at an early transplant generation.

Previous characterization of carcinogen-induced foci and nodules in the rat pancreas have largely used histologic, histochemical, and immunohistochemical methods, although one biochemical study demonstrating variable deficiency of y-glutamyl transpeptidase in isolated nodules has been reported.13 We reported one unsuccessful attempt to transplant and grow foci and nodules.5 These lesions have been divided into acidophilic and basophilic categories on the basis of H&E staining.14 The former have the greater growth potential and are accepted as the precursors of lesions that progress to become carcinomas. Bax et al15 reported that the acidophilic lesions were adenosine triphosphatase (ATPase) positive, whereas basophilic foci were negative. Moore et al16 re-

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B 0

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{.--,.,.L Figure 1. Flow cytometric DNA content of an acinar cell adenoma (A), hypodiploid primary carcinoma (B), and normal pancreas (C). The first small peak is the CRBC internal standard, second is the G,/GO peak, and third is the GJ/M peak.

Table 2. Summa?y of Nodule Growth in SoftAgar No. positive/ % positive No. attempts Type of sample 0/5 0/3 0/3 1/1 3/3 1/1

_l~~~~~~~~~~~~~~

Fluorescence Intensity

ported that acidophilic, ATPase-positive lesions could be stained immunohistochemically using antibody to glutathione-S-transferase placental form. Mori et al17 reported that pancreatic foci and nodules exclude iron, as do enzyme-altered foci in the liver of iron-loaded rats. Morgan et al18 reported that cells of nodules were almost uni-

Nodules, pooled* Adenomas, pooledt Adenomas, single Carcinoma in situ Carcinoma, primary Carcinoma, transplanted

_

0 0

0 100 100 100

Pools consisted of 10 to 15 nodules. Portions of the nodules in two of the pools were examined histologically, whereas the lesions were classified on the basis of size in three pools. t Adenomas in two of the pools were examined histologically and in the third they were classified on the basis of size. All subsequent lesions were examined histologically.

formly mononucleated, whereas 60% to 80% of acinar cells in the normal adult pancreas are binucleate. Binucleation is regarded as a possible indicator of terminal differentiation in the acinar cells.3 None of these special Table 3. Summary of Transplantation Studies. No. Type of sample recipients*

Subcutaneous Transplantst 24 Nodules, n = 8 31 Adenomas, n = 11 24 Carcinoma in situ, n = 9 3 Carcinoma, primary, n = 1 Subcutanenous and Intraperitoneal Transplants 3 Nodules, n = 3 17 Adenomas, n = 13 4 Carcinoma in situ, n = 2 6 Carcinoma, primary, n = 2

No. positive 0 0 1 0 0 0 0 1

* A total of 141 recipients were used in these studies. t Each of these tumors was also placed into a single recipient as a subcapsular renal transplant. All were negative. Each lesion was minced individually and inoculated into one to three recipients.

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Figure 2. Acinar cell adenoma that was transplanted to four recipients. The adenoma isfrom the pancreas ofa male F344 rat that was given corn oil by gavage. None of the explants grew (H&E, X40).

approaches has proven superior to routine histology with regard to identification of a subpopulation of nodules with a high potential to undergo progression to malignancy. Recently the application of a membrane marker of cell polarity has proven useful in subclassifying advanced lesions into categories of apparently -differing malignant potential.19 The latter method may provide a useful approach for subclassifying nodules. Histologic study of large pancreatic nodules and adenomas in experiments of 1 to 2 years' duration often discloses secondary and sometimes even tertiary foci and nodules developing within the primary lesion.3 In liver carcinogenesis, this type of change has been designated as the formation of 'focus in focus' or 'nodule in nodule.'20 The secondary and tertiary foci are defined by further phenotypic changes usually characterized by nuclear enlargement or decreased zymogen content of the acinar cells. Sometimes the derivative lesion shows sufficient anaplasia to support a diagnosis of carcinoma in situ. We anticipated that secondary foci within nodules might more often be aneuploid than the primary nodules and therefore give rise to a second peak in studies of nuclear DNA content by flow cytometry. This was not the case among the lesions studied. The presence of 2N DNA content in four of five CIS and primary carcinomas Table 4. Results of Transfection Experiments Size (mg) Histology Foci/,g DNA CIS 51 0.12 CIS 73 0.13 82 Adenoma 0.02 98 Adenoma 0.05 79 Adenoma 0.01 129 Adenoma 0.03 Each 60-mm culture dish of NIH 3T3 cells was transfected with

20-30 ,ug DNA. The T24 bladder ca cell line (transformation frequency = 0.2) and NIH 3T3 and normal mouse liver (background = 0.02) were included as controls.

suggests that secondary foci could well have normal DNA content even if they represented a further step in progression to malignancy. Overt hyperdiploid DNA content was encountered only in transplantable carcinomas. This could reflect a requirement for progression to a highly malignant phenotype and represent a rare and late event that was not detected among the few primary carcinomas included in this study. Flow cytometric DNA measurements can detect conditions such as polyploidy, aneuploidy, and clonal growth in aneuploid cell populations. These reflect nuclear changes that have been shown to be associated with some aspects of carcinogenesis and malignant progression.21 Polyploidy implies the multiple of chromosome number and DNA content of nuclei and is thought to accompany adaptation to extreme growth conditions. Polyploidy is linked to carcinogenesis, both as a finding in early stages of carcinogenesis and as an effect of carcinogens in several systems. In the present study, it appears that aneuploidy is not a prerequisite for neoplastic growth in the rat pancreas, but that it might be associated with advanced stages of malignant progression. Thus it failed as a marker that would allow early identification of nodules with neoplastic growth potential. Other parameters, including growth in soft agar, transplantability, and the results of transfection, followed a similar general pattern and were uniformly negative in the nodules and adenomas that we studied. Transfection of DNA from CIS yielded more transformants than transfection of DNA from adenomas. Male F344 rats that have not been treated with carcinogens have a varying incidence of acinar cell proliferative growths that are diagnosed as focal hyperplasias and adenomas.4 Histologically, focal hyperplasia is the equivalent of acidophilic foci and nodules as described in azaserine-treated rats, and 'adenoma' designates similar tumors in azaserine-treated and non-carcinogen-treated rats. These tumors are more common in corn-oil-treated animals than in controls fed chow22 and can be demonstrated to be quite common when multiple sections of the pancreas are examined.23 Because the biology of these lesions is not well understood, they were transplanted under the kidney capsule for comparison with the carcinogen-induced lesions. The results indicate that the proliferative lesions associated with corn oil administration are not very aggressive, and we were unable to demonstrate growth in recipients. Because the transplantation of both azaserine-induced and corn oilassociated nodules and adenomas used inbred rat strains, we do not believe that immunologic responses hindered growth. Rather, we think that our results indicate that these lesions have limited growth potential and a nonmalignant phenotype.

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Specific oncogenes that are associated with pancreatic carcinoma in the rat have not yet been identified. We recently noted increased expression of c-myc, c-raf-1, and c-K-ras in nodules, adenomas, and carcinomas,24 but it is not clear if such expression reflects a critical initiating event or is secondary. Levels of c-myc expression relative to normal rat pancreas were twofold to ninefold increased in three adenomas, fourfold to ninefold increased in two primary carcinomas, and 18- to 58-fold increased in five transplanted carcinomas based on densitometry of Northern blots. For c-raf-1, the comparable levels were 1- to 12-, 7- to 11-, and 14- to 45-fold increases. c-K-ras expression could not be measured in normal pancreas, so comparable ratios are not available; however expression clearly was elevated in transplantable carcinomas. Expression of c-K-ras was demonstrated in only one of three adenomas examined. We have sought and failed to find mutated c-K-ras in DNA from rat pancreatic carcinomas and adenomas using the polymerase chain reaction and oligomer hybridization to identify specific mutations.25 Thus the transformed foci of 3T3 cells are presumed to contain an activated oncogene or to be overexpressing cellular oncogenes that we have not identified. As activation of specific oncogenes or inactivation of tumor suppressor genes, eg, p53, are identified in human or animal pancreatic carcinomas,26 it will be appropriate to extend the evaluation of such genes to the rat model. At the time these studies were completed, the principal focus in human pancreatic carcinomas was on ras mutations. In summary, cells of nodules and adenomas have low growth potential and lack critical phenotypic characteristics of transformed malignant cells. We have not defined phenotypic traits or changes in cellular ondog-e-s that identify subtypes associated with high growth potential other than anaplastic cellular changes that are the basis for a histologic diagnosis of carcinoma. Some, but not all, carcinomas were demonstrated to have aneuploid nuclear DNA content, to grow in soft agar, and to be tumorigenic on transplantation. No basis was defined, however, for distinguishing between the categories of large nodule and adenoma except for size.

Acknowledgments The authors thank Nancy Bigelow for technical assistance with flow cytometry and Karen Doran for assistance in animal care. The transplantation studies of nodules from oil-gavaged rats were done in Dr. Boorman's laboratory at NIEHS.

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and Diseases. Edited by VLW Go. New York, Raven Press, 1986, pp 443-458 2. Longnecker DS: The azaserine-induced model of pancreatic carcinogenesis in rats, Experimental Pancreatic Carcinogenesis. Edited by DG Scarpelli, JK Reddy, DS Longnecker. Boca Raton, FL, CRC Press, 1987, pp 11 7-130 3. Longnecker DS: Interface between adaptive and neoplastic growth in the pancreas. Gut 1987, 28(suppl):253-258 4. Eustis SL, Boorman GA: Proliferative lesions of the exocrine pancreas: Relationship to com oil gavage in the national toxicology program. JNCI 1985, 75:1067-1073 5. Longnecker DS, Lilja HS, French JI, Kuhlmann E, Noll WW: Transplantation of azasenne-induced carcinomas of pancreas in rats. Cancer Lett 1979, 7:197-202 6. Sumi C, Brnck-Johnsen T, Longnecker DS: Inhibition of a transplantable pancreatic carcinoma by castration and estradiol in rats. Cancer Res 1989, 49:6687-6692 7. Vindelov LL, Christensen IbJ, Nissen NI: A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983, 3:323-327 8. Perucho M, Goldfarb M, Shimizu K, Lama C, Fogh J, Wigler M: Human-tumor-derived cell lines contain common and different transforming genes. Cell 1981, 27:467-476 9. Southem PJ, Berg P: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1982, 1:327-341 10. Fasano 0, Bimbaum 0, Edlund L, Fogh J, Wigler M: New human transforming genes detected by a tumorigenicity assay. Mol Cell Biol 1984, 4:1695-1705 11. Chomzynski P, Qasba PK: Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun 1984, 122:340-344 12. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983,132:6-13, and 1984,137:26667 13. Faribault G, Wiebkin P, Hamilton JW, Longnecker DS, CurpheyTJ: y-Glutamyltransferase activity in atypical acinar cell nodules of rat pancreas. Toxicol AppI Pharmacol 1987,

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by azaserine. Carcinogenesis 1986, 7:457-462 16. Moore MA, Makino T, Tsuchida S, Sato K, Ichihara A, Amelizad A, Oesch F, Konishi Y: Altered drug metabolizing potential of acinar cell lesions induced in rat pancreas by hydroxyaminoquinoline 1-oxide. Carcinogenesis 1987, 8:1089-1094 17. Mori H, Tanaka T, Takahasi M, Williams G: Exclusion of cellular iron and reduced gamma-glutamyl transpeptidase activity in rat pancreas acinar cell hyperplastic nodules and adenomas induced by azaserine. Gann 1983, 74:497-501

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18. Morgan RGH, Schaeffer BK, Longnecker DS: Size and number of nuclei differ in normal and neoplastic acinar cells from rat pancreas. Pancreas 1986, 1:37-43 19. Takiyama Y, Woutersen RA, Pour PM: Ulex Europaeus-l: A marker for differentiation of (pre)cancerous lesions induced in the rat pancreas by azaserine. Carcinogenesis 1988, 9:2087-2092 20. Scherer E: Neoplastic progression in experimental hepatocarcinogenesis. Biochim Biophys Acta 1984, 738:219-236 21. Merkel DE, McGuire WL: Ploidy, proliferative activity and prognosis. DNA flow cytometry of solid tumors. Cancer 1990, 65:1194-1205 22. Haseman JK, Huff, JE, Rao GN, Amold JE, Boorman GA, McConnell EE: Neoplasms observed in untreated and corn

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oil gavage control groups of F344/N rats and (C57BU6N x C3H/HeN)F1 (B6C3F,) mice. JNCI 1985, 75:975-984 Boorman GA, Banas DA, Eustis SL, Haseman JK: Proliferative exocrine pancreatic lesions in rats. The effect of sample size on the incidence of lesions. Toxicol Pathol 1987, 15:451-456 Silverman J, Kuhlmann E, Yager J, Zurlo J, Longnecker D: Oncogene expression in azaserine induced pancreatic carcinomas in rats. Proc Am Assoc Cancer Res 1990, 31:311 Schaeffer BK, Zurlo J, Longnecker DS: Activation of c-K-ras not detectable in adenomas or adenocarcinomas arising in rat pancreas. Molecular Carcinogenesis 1990, 3:165-170 Perucho M, Capella G, Shibata D: Oncogene activation in human pancreatic adenocarcinoma. Proc Am Assoc Cancer Res 1990, 31:478-479