Effects of dimethylnitrosamine on organ-cultured adult human pancreas

Portions of adult human pancreas from 20 donors were organ-cultured in a chemically defined medium in the absence or presence of DMNA for up to 12 weeks. In the absence of DMNA, necrosis of some acini occurred during the first week, while some clusters of well-preserved acini were maintained for up to 3 weeks. Proliferation of the epithelial linings of main and smaller ducts and ductules was noted during the first 2 weeks of culture. Ductal epitheliums thereafter showed some degeneration but remained viable during the 12 weeks of culture. In contrast to controls, the DMNA-treated explants showed better preservation of both acinar and ductal cells. DMNA induced both ductal hyperplasia and atypia of the epithelial linings of main ducts, smaller ducts, and ductules within 6 weeks, and carcinoma by the tenth week of culture. At the end of the first week cells devoid of zymogen within the acinar complex proliferated and progressively replaced necrotic cells. During the ninth and tenth weeks, foci of atypical cells developed among these cells. Cells derived from 10-week-old DMNA-treated explants produced multiple nodules of carcinoma when injected subcutaneously into nude mice.     

What's new in in vitro studies of exocrine pancreatic cell injury?

Exocrine pancreas in vitro models are useful for the study of pancreatic differentiation, secretion mechanisms, cell injury, and lysosomal processing of secretory product. Syrian hamster pancreas in explant organ culture undergoes a series of morphologic changes which parallel in vitro acinar cell injury, differentiation, and phenotypic alteration. Within 48 hours, the cultured acinar cells show morphologic evidence of sublethal cell injury. Autophagy and crinophagy are particularly striking. The autophagic processes can be inhibited by the addition of the protein synthesis inhibitor cycloheximide or by culture at lowered temperatures (20 degrees C). Acinar cells lethally damaged show pyknotic nuclei, high amplitude swelling, and necrosis. Approximately 25% of each explant is viable after 72 hr in culture and the viability remains constant at 25-35% for up to 60 days of culture. The morphological changes of the explants are consistent with many of the features of pancreatitis and carcinoma of the exocrine pancreas. There is an increase in the ductal elements and a decrease in acini over time in culture. This may be due to: (a) an increased replication of ductal epithelial cells concomitant with necrosis of acinar epithelial cells and/or (b) phenotypic alteration of acinar cells to ductal cells. Acinar cell necrosis and phenotypic alterations may in part be due to the activation of lysosomal degradation pathways. Processes which inhibit lysosomal activation proved protective against these alterations, while processes which promote zymogen activation were deleterious.     

Pancreatic growth and cell turnover in the rat fed raw soya flour.

Growth and differentiation of the pancreatic acinar cell was studied in rats fed raw soya flour (RSF) for up to a year. A second group of rats were fed a control diet. After 1 week of RSF feeding there was a 200% increase in tissue RNA and weight, indicating initial hypertrophy, which was maintained for the 1-year study period. By the second week and over the remainder of the period studied there was also a marked increase in total DNA, suggesting hyperplasia. Cell turnover, as measured by the rate of incorporation of 3H-thymidine into pancreatic DNA, was significantly higher in RSF-fed animals only from the second to fourth weeks; it then returned to control values. Autoradiography showed an 18-fold increase in duct cell labeling at the end of the first week and an 11-fold increase by the end of the second week. Acinar cell labeling doubled from the second to the twelfth week. These studies confirm previous reports that RSF produces pancreatic hypertrophy and hyperplasia. They furthermore show that there is initially marked stimulation of DNA synthesis in the duct cell compartment. The results suggest that cells with the morphologic characteristics of duct cells may be the precursors of acinar cells in hyperplastic pancreatic tissue.     

The development of the granular convoluted duct in the rat submandibular gland.

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given 3-H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 X 10-6) at four weeks to 26% (68 X 10-6) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.     

Proliferation of normal breast epithelial cells as shown by in vivo labeling with bromodeoxyuridine.

The proliferative activity of normal acinar and ductal breast epithelial cells was studied by in vivo labeling with 5-bromodeoxyuridine (BrdUrd) in 26 cases with concurrent breast carcinoma. The BrdUrd-labeled cells were recognized in histologic sections of paraffin-embedded tissue, using an anti-BrdUrd antibody and an immunoperoxidase reaction. The percentage of BrdUrd-labeled cells showed great variability for both acinar (0% to 2.66%; mean, 0.70%; standard deviation [SD], 0.80%) and ductal cells (0% to 1.99%; mean, 0.51%; SD, 0.57%). The fraction of proliferating epithelial cells declined with the age of the patients and was significantly higher in premenopausal women (1.16% +/- 0.85% for acinar and 0.94% +/- 0.60% for ductal cells) as compared with the postmenopausal women (0.27% +/- 0.46% for acinar and 0.17% +/- 0.22% for ductal cells), P less than 0.01 for acinar and P less than 0.001 for ductal cells, respectively. In some patients, great variability in distribution of proliferating acinar and ductal cells among different lobules and ducts was observed. No difference was found in the number of proliferating acinar and ductal cells situated near or far from their corresponding tumors. No correlation was seen between cell proliferation of normal acinar or ductal cells and cell proliferation of the respective tumors.     

The development of the granular convoluted duct in the rat submandibular gland

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given ³H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 × 10⁶) at four weeks to 26% (68 × 10⁶) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.     

Long-term organ culture of embryonic rat pancreas in a chemically defined medium.

Embryonic rat pancreas anlagen have been grown in a chemically defined medium, subdivided biweekly and recultured for a total time of 10 weeks. The total mass increment during this time was in excess of 1000-fold. Samples were removed for light and electron microscope examination and for periodic measurement of enzymatic activity. Morphogenesis and cytodifferentiation occurred, peripheral outgrowth of epithelial buds was followed by the formation of interconnecting tubular structures and, eventually, by the appearance of distinctive acinar cells with zymogen granules. Mitotic figures became conspicuous at the periphery of explants within a day after each subdivision resulting in the formation of new tubular structures and acini. In general, the central area of the explants presented more mature acini with zymogen granules than was manifested at the periphery. The enzymatic activities of amylase, lipase, and chymotrypsin developed maximally during the first week of culture, reached a plateau level by the second week, and remained at a relatively constant level throughout the 10-week culture period.     

An in vitro model of pancreas carcinogenesis: two-stage MNU--TPA effect.

Organ-cultured embryonic rat pancreata were exposed to either single or multiple doses of methylnitrosourea (MNU), a single dose of MNU followed by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or TPA alone and cultured for up to 6 weeks. Both single and multiple doses of MNU caused similar alterations during the first 10 days; ie, for 4 days the explants grew and differentiated as untreated explants, forming acini and ductules; thereafter the presence of MNU induced ductular proliferation and hyperplasia. Explants exposed to a single dose of MNU failed to proliferate beyond the 10th day of culture, showed progressive cell necrosis, and became almost completely necrotic in 6 weeks. Cells prepared from these explants on Day 10 and injected subcutaneously into nude mice also failed to grow and degenerated after 2 weeks. Multiple doses of MNU in vitro, however, produced further proliferation with an atypical cribriform pattern by the 15th day. In the absence of MNU, treatment with TPA alone had no histologic effect; but TPA treatment after a single dose of MNU promoted abnormal growth similar to that produced by multiple doses of MNU. Cells prepared from 10-day explants treated with a single dose of MNU followed by TPA grew subcutaneously in nude mice and formed nodules of atypical growth within 2 weeks. This system constitutes a simple model of short-latency chemical carcinogenesis.     

Early lesions of pancreatic ductal carcinoma in the hamster model.

Syrian golden hamsters were treated weekly with 10 mg/kg body weight N-nitrosobis (2-oxopropyl) amine for life (Group 1) or 6 weeks and were sacrificed at biweekly intervals from 2 weeks (Group 1) and 8 weeks (Group 2) after initiation of the experiment. The pancreas was examined in step sections, and the sequential alterations noted for each interval were recorded. Lesions were found in intrapancreatic and extrapancreatic ducts. Equivalent alterations consisting of hyperplasia, metaplasia, atypia, and lesions characteristic of carcinoma in situ developed ubiquitously and simultaneously in pancreatic ducts of different sizes and in ductules, but not in acinar cells. Among the most significant findings were intrainsular ductular formations, their proliferation, and sequential malignant alteration comparable to the involved preexisting ductules. Differences between the two experimental groups were of a quantitative rather than qualitative nature. The incidence and multiplicity of neoplastic lesions at each interval according to group, sex, and anatomic locations of adenocarcinomas are outlined. Predilected areas for some lesions were found. Results indicate a common origin of all induced tumors from a pluripotent cell populating the pancreatic ductal system.     

Hepatocytic differentiation of cultured rat pancreatic ductal epithelial cells after in vivo implantation.

We have investigated the differentiation potential of propagable cultured rat pancreatic duct epithelial cells after in vivo implantation in isogeneic Fischer-344 rats. Cells genetically labeled with Escherichia coli beta-galactosidase (lacZ) reporter gene were embedded in a mixture of collagen and Matrigel (basement membrane matrix) and implanted either subcutaneously or intraperitoneally. Tissues from the two locations were harvested 4 to 8 weeks later. The great majority of the lacZ-labeled epithelial cells colonizing both sites phenotypically resembled hepatocytes, although they demonstrated different degrees of hepatocytic differentiation. Less than 5% of lacZ-labeled cells formed ductular structures. The hepatocyte-like cells from the subcutaneous implantation site expressed mixed phenotypes of both hepatocyte and ductal cell, including the expression of alpha-fetoprotein, tyrosine amino-transferase, gamma-glutamyl transpeptidase, carbonic anhydrase II, and cytokeratin 19. In contrast, the hepatocyte-like cells colonizing the mesentery showed the phenotype of mature hepatocytes, including an abundant glycogen storage and a lack of alpha-fetoprotein and carbonic anhydrase II expressions. Neither acinar cell nor endocrine differentiation was seen. These findings demonstrate that pancreatic ductal cells can be the progenitor cell for transdifferentiated hepatocytes.     

MCF10AT: a model for the evolution of cancer from proliferative breast disease.

A human cell line (MCF10A) originated from spontaneous immortalization of breast epithelial cells obtained from a patient with fibrocystic disease. MCF10A cells do not survive in vivo in immunodeficient mice. However, T24 c-Ha-ras oncogene-transfected MCF10A cells (MCF10AT) form small nodules in nude/beige mice that persist for at least 1 year and sporadically progress to carcinomas. By reestablishing cells in tissue culture from one of the carcinomas, a cell line designated MCF10AT1 was derived that forms simple ducts when transplanted in Matrigel into immunodeficient mice. With time in vivo, the epithelium becomes proliferative and a cribriform pattern develops within the xenografts. A significant number progress to lesions resembling atypical hyperplasia and carcinoma in situ in women, and approximately 25% progress to invasive carcinomas with various types of differentiation including glandular, squamous, and undifferentiated. Cells have been established in culture from lesions representing successive transplant generations. With each generation, cells are somewhat more likely to progress to high risk lesions resembling human proliferative breast disease. Although the incidence of invasive carcinoma remained fairly constant at 20 to 25%, the frequency of nodules showing proliferative breast disease rose from 23% in the first transplant generation to 56% in the fourth transplant generation.     

Acinic cell-like differentiation in invasive ductal carcinoma and in ductal hyperplasia of the breast--report of two cases

Described are two epithelial lesions of the breast displaying extremely rare, widespread acinic cell-like differentiation (metaplasia). Two women, 70 and 40-year-old, one with invasive ductal papillocarcinoma, the other one with conventional intraductal hyperplasia without atypia, both demonstrated massive diffuse, PAS positive, granular eosinophilic transformation of the cell cytoplasm. This unusual cell appearance closely simulated acinar cells in normal serous salivary gland/acinic cell carcinoma or Paneth cells. Both extensive expression of lysozyme and finding of numerous zymogen granules ultrastructurally confirmed the acinic cell-like fenotype. Discussed is differential diagnosis of the breast neoplasm containing overt eosinophilic and granular cytoplasm. Reviewing literature and comparing our unique finding of unusual salivary-type differentiation in conventional ductal hyperplasia of the breast, biologic implications are considered.     

Multiple atypical acinar cell nodules of the pancreas

Although acinar cell nodules of the pancreas have been described in rats given carcinogenic chemicals, similar nodules have not been reported in humans. This report describes two cases of nodular acinar cell lesions in the pancreas in patients who died of insulin secreting islet cell adenoma and of bronchogenic carcinoma. The lesions consisted of multiple nodules that were well demarcated from the surrounding acinar tissue and were composed of zymogen granule containing acinar cells with a pale to pink cytoplasm. The significance of these atypical acinar cell nodules in regard to their being possible precursor lesions of acinar cell carcinoma of the pancreas is discussed.     

Derivation of ductlike cell lines from a transplantable acinar cell carcinoma of the rat pancreas.

Two cell lines were derived from a transplantable acinar cell carcinoma that had been established from a primary carcinoma of the pancreas in an azaserine-treated Lewis rat. The cultured tumor cells initially produced amylase, but production of exocrine enzymes ceased after 1-2 weeks in culture. The cultured cells were tumorigenic in Lewis rats, and one line produced solid tumors composed of ductlike structures surrounded by dense fibrous tissue. The second cell line produced partially solid and partially cystic tumors with a mixed phenotype of squamous, mucinous, and glandular areas when it grew in vivo following regrafting. Both cell lines lost structural and immunohistochemical acinar cell markers while acquiring duct cell markers during culture and regrafting. These studies provide strong support for the hypothesis that ductlike carcinomas can arise from neoplastic pancreatic acinar cells in rats.     

Double neuroendocrine ductal carcinomas in situ coexisting with a background of diffuse idiopathic neuroendocrine cell hyperplasia of breast: a case report and hypothesis of neuroendocrine tumor development

This article reports the case of a 72-year-old woman with two nodules of neuroendocrine (NE) ductal carcinoma in situ coexistent with a background of NE cell hyperplasia. Both tumors, 15 and 3 mm in size, were incidentally revealed on computed tomography without any apparent clinical symptoms. The tumors showed similar histological features, and more than 50% of the tumor cells patchily expressed NE markers, such as chromogranin A, synaptophysin, CD56, and somatostatin receptor type 2. The surrounding nontumor ductal cells also showed spotty or linear positivity for NE markers in contrast to the cells of normal atrophic breasts, which rarely present with NE cells. Moreover, focal mucin production was also observed in the peripheral ducts. It is hypothesized that idiopathic breast NE cell hyperplasia with multiple small nests of NE cells may extend to form a true mass of NE neoplasms.     

Modulation of carbohydrate residues in regenerative nodules and neoplasms of canine and feline pancreas.

The glycoconjugates of regenerative acinar cells, acinic cell carcinomas, islet cell tumors, and normal canine and feline pancreas were studied. The authors used biotinylated lectins as probes and avidin-biotin-peroxidase complex as visualant to identify and to compare the distribution of carbohydrate residues on paraffin sections from 74 cases. The findings demonstrate a difference in the staining pattern between normal acinar, islet, and ductal cells in each species and small differences in the staining pattern between the species. It is shown that in nodules of regenerative acinar cells and acinic cell carcinomas there is an increased staining intensity with Concanavalia ensiformis agglutinin, Ricinus communis agglutinin-I, and wheat germ agglutinin. The pattern of lectin staining in regenerative cells and malignant acinar cells reflects the degree of cellular differentiation. Intensive apical staining characterizes a higher degree of differentiation, while dispersed staining is a major feature of poor differentiation. These findings suggest that malignant transformation of pancreatic acinar cells is associated with enhanced expression of glycoconjugates, which resembles that seen in a normal immature acinar cells.     

Adequate histologic sampling of breast core needle biopsies

OBJECTIVE: To determine the degree of histologic sampling necessary for adequate examination of breast core needle biopsy specimens. DESIGN: The results of all breast core needle biopsies (11 and 14 gauge) with a diagnosis of atypical small acinar proliferation or atypical ductal hyperplasia and subsequent excisional biopsies, for a 50-month period were reviewed. Blocks of all cores were sectioned entirely in 8 slides to determine the amount of sectioning needed to detect these foci, and the results were correlated with those from the excision specimen. SETTING: Large community hospital practice. RESULTS: Of 3026 cases, 216 (7.1%) were diagnosed as atypical ductal hyperplasia or atypia not otherwise specified. Subsequent resections were available in 105 (49%) cases, and after review, 95 (92%) qualified as atypical ductal hyperplasia and 2 were determined to be atypical small acinar proliferations. The 2 small acinar proliferations were first detected on the second and fourth slides. Of the atypical ductal hyperplasia cases, 43% were detected on the first slide, 17% on the second, 23% on the third, 8% on the fourth, and 8% on the fifth. No lesions were initially detected after this level. Ductal carcinoma in situ was detected in the excision specimens from 1 case each of those detected initially on the fourth and fifth slides. CONCLUSION: Five sections of breast core needle biopsy specimens are necessary to ensure that all atypical small acinar proliferations and atypical ductal hyperplasia lesions are sampled.     

Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas.

Experiments have been performed to define conditions for the primary culture of human exocrine pancreas, as a first step towards molecular reconstruction experiments of pancreatic neoplasia. Normal human exocrine pancreas was digested using collagenase and dispase and the resulting cellular aggregates were cultured in vitro. The phenotype of the digested pancreatic cells was almost exclusively acinar (amylase-positive, keratin 19 and mucin antigens-negative), yet within 4 days of culture the cells had taken on a ductal phenotype (amylase-negative, keratin 19 and mucin antigens-positive). The kinetics of these observations exclude the possibility of overgrowth of the acinar population by a ductal sub-population, and selective adherence is excluded by examination of those cells that do not adhere, which are representative of the initiating population. We interpret these data as indicating that, under the conditions of culture, the acinar cell phenotype is not stable and can transdifferentiate to a ductal phenotype. Taken together with recent data from transgenic animals, this in vitro observation has possible implications for our view of the pathogenesis of pancreatic neoplasia.     

TAZ inhibits acinar cell differentiation but promotes immature ductal cell proliferation in adult mouse salivary glands

There are currently no treatments for salivary gland diseases, making it vital to understand signaling mechanisms operating in acinar and ductal cells so as to develop regenerative therapies. To date, little work has focused on elucidating the signaling cascades controlling the differentiation of these cell types in adult mammals. To analyze the function of the Hippo-TAZ/YAP1 pathway in adult mouse salivary glands, we generated adMOB1DKO mice in which both MOB1A and MOB1B were TAM-inducibly deleted when the animals were adults. Three weeks after TAM treatment, adMOB1DKO mice exhibited smaller submandibular glands (SMGs) than controls with a decreased number of acinar cells and an increased number of immature dysplastic ductal cells. The mutants suffered from reduced saliva production accompanied by mild inflammatory cell infiltration and fibrosis in SMGs, similar to the Sjogren's syndrome. MOB1-deficient acinar cells showed normal proliferation and apoptosis but decreased differentiation, leading to an increase in acinar/ductal bilineage progenitor cells. These changes were TAZ-dependent but YAP1-independent. Biochemically, MOB1-deficient salivary epithelial cells showed activation of the TAZ/YAP1 and β-catenin in ductal cells, but reduced SOX2 and SOX10 expression in acinar cells. Thus, Hippo-TAZ signaling is critical for proper ductal and acinar cell differentiation and function i n adult mice.     

Transforming growth factor alpha in epithelial proliferative diseases of the breast.

AIMS: To determine at what stage there is increased expression of transforming growth factor alpha (TGF alpha) in preneoplastic diseases of the breast and to determine if this would assist in the histological diagnosis of different intraduct epithelial proliferations. METHODS: Specimens were retrieved from the archives of 17 cases of ductal hyperplasia, six cases of atypical ductal hyperplasia and 13 cases of ductal carcinoma in situ together with 12 'normal' breast biopsy specimens. Sections were stained immunohistochemically for TGF alpha. The staining was assessed semi-quantitatively taking into account both the staining intensity and the proportion of cells stained. RESULTS: Minimal expression of TGF alpha was observed in normal breast tissue. Increased levels of expression were seen in ductal hyperplasia, atypical ductal hyperplasia, and ductal carcinoma in situ. Increased levels of expression of TGF alpha were also found in morphologically normal ducts immediately adjacent to areas of intraduct epithelial proliferation. CONCLUSION: Increased expression of TGF alpha occurs in the early stages of intraduct epithelial proliferation and will not help the histopathologist distinguish atypical ductal hyperplasia from either ductal hyperplasia or ductal carcinoma in situ. The molecular changes within a cell may precede the morphological changes observed by light microscopy thereby reflecting the biological potential of the epithelium.