Telepathology in India.

Telepathology is the most recent addition to the pathologist's diagnostic tools. It is the acquisition of macroscopic and microscopic images for electronic transmission for diagnosis, consultation and/or education. With the addition of the personal computer at the pathologist's desktop, the stage has been set for one of the greatest advantages the Internet has to offer. Telepathology in India is in infancy, and we at PathoIndia (www.Pathoindia.com) have started a series of publication images from interesting cases in the form of weekly quiz. After cases are published, hundreds of pathologists from around the world are invited by e-mail to send in their diagnosis and comments. The responses to this quiz suggest that telepathology is catching on in the pathology community. Another intention of this series is to identify and select qualified international and Indian pathologists who would be willing to help colleagues from India requesting second opinions online.

Relative sensitivity of undifferentiated and cyclic adenosine 3',5'-monophosphate-induced differentiated neuroblastoma cells to cyclosporin A: potential role of beta-amyloid and ubiquitin in neurotoxicity.

Cyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3',5'-monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E1, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or beta-amyloid peptide (amino acids 1-14) (Abeta1-14); however, it enhanced the intensity of staining for both ubiquitin and Abeta in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44-63) (APP44-66) did not change in any treated group, suggesting that the increase in Abeta staining is due to increased processing of APP to Abeta. We propose that one of the mechanisms of cyclosporin A-induced neurotoxicity involves increased levels of Abeta and ubiquitin.

Aluminum increases levels of beta-amyloid and ubiquitin in neuroblastoma but not in glioma cells.

Several epidemiological studies suggest the involvement of aluminum (Al) in the pathogenesis of Alzheimer's disease (AD). There is an increase in the levels of Abeta and ubiquitin in the pathological lesions of AD. Therefore, we have investigated whether aluminum (Al) treatment alters the levels of Abeta and ubiquitin in murine neuroblastoma (NBP2) and rat glioma (C-6) cell cultures. At a low concentration (10 microM), aluminum sulfate stimulated the level of immunoreactive Abeta and ubiquitin in NBP2 cells without changing the levels of the amyloid precursor protein (APP). However, at higher concentrations (100 and 500 microM), aluminum failed to elicit any significant effect on beta-amyloid, whereas ubiquitin levels continued to increase. No changes in the Abeta and ubiquitin content were found in the C-6 glioma cells following treatment with Al at any of the concentrations tested. Exposure of cells to aluminum salts did not alter the rate of proliferation in either of the two cell lines. These data suggest that one of the mechanisms by which Al may play a role in AD is by promoting the formation of Abeta and ubiquitin in neurons.

Adenosine 3',5'-cyclic monophosphate increases processing of amyloid precursor protein (APP) to beta-amyloid in neuroblastoma cells without changing APP levels or expression of APP mRNA.

Beta-Amyloid (Abeta), a 39-43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimer's disease (AD); and therefore, a role of Abeta in neuronal degeneration has been proposed. The factors which regulate the levels of Abeta have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3', 5'-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of Abeta levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of Abeta immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to Abeta increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or Abeta.

Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture.

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.

Prostaglandins as putative neurotoxins in Alzheimer's disease.

Chronic inflammatory reactions in the brain appear to be one of the primary etiological factors in the pathogenesis of Alzheimer's disease (AD). This is supported by the fact that the secretory products of inflammatory reactions, which include cytokines, complement proteins, adhesion molecules, and free radicals, are neurotoxic. We have recently reported that prostaglandins (PGs), which are also released during inflammatory reactions, cause rapid degenerative changes in differentiated murine neuroblastoma cells (NB) in culture. PGA1 is more effective than PGE1. Similar observations were made in a primary culture of fetal rat hippocampal cells. Epidemiological and clinical studies on AD also support the involvement of PGs in neuronal degeneration. Thus, we propose a hypothesis that PGs are one of the major extracellular signals that initiate neuronal degeneration, which is mediated by intracellular signals such as the beta-amyloid peptide (Abeta) and ubiquitin, since the levels of these proteins are increased by PG treatment. We further suggest that adenosine 3', 5'-cyclic monophosphate (cAMP) is one of the factors that regulate the levels of both Abeta and ubiquitin in NB cells. Increases in the level of Abeta in NB cells following an elevation of intracellular cAMP levels appear to be due to an increase in the rate of processing of the amyloid precursor protein (APP) rather than an increase in the expression of APP. The mechanisms underlying Abeta-induced neuronal degeneration have been under intense investigation, and several mechanisms of action have been proposed. We postulate that PG-induced elevation of Abeta may lead to an increased binding of Abeta to the 20S proteasome, resulting in a reduction of 20S proteasome-mediated degradation of ubiquitin-conjugated proteins. This is predicted to lead to an increase in an accumulation of abnormal proteins, which ultimately contribute to neuronal degeneration and death. Based on our hypothesis and on studies published by others, we propose that a combination of nonsteroidal anti-inflammatory drugs, which inhibit the synthesis of PGs, and antioxidant vitamins, which quench free radicals and both of which have been recently reported to be of some value in AD treatment when used-individually, may be much more effective in the prevention and treatment of AD than the individual agents alone.

Efficacy of grafted immortalized dopamine neurons in an animal model of parkinsonism: a review.

Dopamine (DA) deficiency is one of the primary lesions in the pathogenesis of Parkinson disease (PD). Because of long-term toxicity of L-DOPA therapy, the grafting of fetal mesencephalic tissue containing dopamine neurons or homogeneous populations of DA neurons into striatum appears to be rational. Fetal tissue transplants have many problems which include legal (in some countries), ethical, paucity of tissue availability, heterogenicity of cell populations, and the presence of antigen-presenting cells that are responsible for rejection of allogeneic grafts. In order to resolve the above problems, we have established immortalized DA neurons from fetal rat mesencephalon by inserting the large T-antigen (LTa) gene of the SV40 virus into the cells. A clone of DA neurons (1RB3AN27) was isolated, characterized, and tested in 6-hydroxydopamine (6-OHDA)-lesioned rats (a model of PD). These cells divided with a doubling time of about 26 h, expressed the LTa gene, and contained the tyrosine hydroxylase and dopamine transporter proteins and their respective mRNAs, which became elevated upon differentiation. These cells were nontumorigenic and nonimmunogenic and improved the symptoms of neurological deficits (methamphetamine-induced rotation) in 6-OHDA-lesioned rats. The differentiated DA neurons were more effective than undifferentiated ones. These studies suggest that immortalized DA neurons generated in vitro by LTa gene insertion may be used in transplant therapy without fear of tumor formation or rejection.

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and beta-amyloid.

Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer's disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE1-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA1 was more effective than PGE1 in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and beta-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and beta-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and beta-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and beta-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.

Brain contains inhibiting factors specific to the large T-antigen gene.

SV40 large T-antigen (LTa) gene-induced immortalized rat dopamine-producing nerve cells (IRB3AN27), which produce LTa protein and divide in vitro, do not divide and do not produce LTa protein when transplanted into striatum of adult rats. This suggests the presence of LTa gene-inhibiting factors in brain. Here we report that rat brain soluble fraction (SF) contains factors which specifically inhibit LTa gene activity in vitro. The brain SF inhibited LTa protein levels and the growth of IRB3AN27 cells and 2RSG cells (LTa gene-induced immortalized rat parotid acinar cells) in vitro, but it stimulated the growth of spontaneously immortalized human parotid acinar cells (2HPC8) and had no effect on the proliferation of murine neuroblastoma cells (NBP2) and rat glioma cells (C-6) in culture. In contrast, the liver SF inhibited the growth of all cell lines tested at varying degrees and thus lacked specificity with respect to LTa gene activity. The presence of specific LTa gene-inhibiting factors in the brain and general tumor growth-inhibiting factors in the liver may provide some of the mechanisms of protection against in vivo carcinogenesis.

Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis.

Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.

Fine needle aspiration cytology of intramammary lymph nodes in an HIV-1-positive patient. A case report.

BACKGROUND: Although rare, intramammary lymph nodes can occur in any quadrant of the breast and display a variety of pathologic conditions. Intramammary lymph nodes may be detected by routine clinical examination, mammography or ultrasound or during gross surgical pathology examination of breast specimens. CASE: A 42-year-old, black woman, HIV-1 positive, presented with bilateral mirror-image breast masses. Fine needle aspiration cytology ruled out the presence of malignancy and confirmed the diagnosis of benign, reactive intramammary lymphadenopathy. CONCLUSION: Clinicians and pathologists should be alert to the existence and potential importance of intramammary lymph nodes in the differential diagnosis of a breast mass in HIV-1-positive patients.

Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations.

Rat dopamine-producing nerve cells (1RB3AN27) and rat parotid acinar cells (2RSG) were immortalized by insertion of simian virus 40 (SV40) large T-antigen gene (LTa). Both of these cells divided and produced nuclear LTa in vitro. In order to assess the relationship between cell proliferation and expression of LTa in vivo, immortalized dopamine-producing nerve cells and parotid cells were grafted into the striatum and parotid gland of adult Sprague-Dawley rats, respectively. Grafted cells exhibited nuclear LTa at 1 day but not at 7 and 30 days after transplantation. At 30 days after transplantation, no tumor was found, and there was no evidence of cell division as determined by H and E staining. When the striatal areas containing the grafts were cultured, these cells did not express LTa at 4 days after plating; however, after 3 weeks, when most host cells were eliminated, the cultured grafted cells expressed LTa. After 3 months of culturing, only cells exhibiting LTa were present. These cells had the same morphology and divided with the same doubling time as 1RB3AN27 cells before grafting. Results suggest the presence of a LTa-inhibiting factor in vivo, and support the hypothesis that the expression of LTa is directly linked with proliferation of immortalized cells.

Brodifacoum intoxication with marijuana smoking.

We report the case of a 17-year-old boy with a significant history of drug and alcohol abuse, which included smoking marijuana mixed with brodifacoum. As a consequence, the patient developed a prolonged coagulopathy that persisted for more than 1 year. To our knowledge, this is the first case reported in the literature in which super-warfarin intoxication has been associated with marijuana smoking. This report should increase the awareness of pathologists and clinicians when examining a patient with a history of drug abuse who exhibits persistent vitamin K1-dependent coagulopathy.

Molar pregnancy in a 60-year-old woman.

Molar pregnancy has not been reported in women past the age of 57 in the English literature. This report reviews a 60-year-old woman who presented with irregular bleeding and was diagnosed as having a complete hydatidiform mole. The patient underwent hysterectomy and had spontaneous return of her human chorionic gonadotropin levels to normal. Diagnosis in this age group depends on a high level of suspicion, and hysterectomy should be considered due to the high risk of postmolar gestational trophoblastic tumor after suction uterine evacuation.

Transplantation of immortalized, nontumorigenic parotid acinar cells into the allogeneic rat parotid gland and oral submucosa.

The recent establishment of an immortalized clonal cell line of rat parotid acinar cells (2RSG) by transfecting isoproterenol-stimulated parotid cells with a plasmid vector, pSV3neo which carries the large T-antigen gene from SV40 virus, afforded the opportunity to develop a model for parotid acinar cell transplantation. Single cell suspensions of 2RSG cells labeled with a fluorescent tracer, DiI, were injected into the parotid gland or oral submucosa of allogeneic adult rats. The grafted cells survived and were functionally viable for at least 30 days. Histological sections revealed no evidence of infiltration of leukocytes or lymphocytes. Grafted cells did not form tumors. Results suggest that allogeneic parotid acinar cell transplantation is a feasible technique in the animal model.

Increased expression of ubiquitin during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

The role of ubiquitin in proliferation and differentiation of nerve cells has not been studied. An elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) in neuroblastoma cells induces terminal differentiation in these cells. Therefore, in this study we investigated the changes in the level and subcellular distribution of ubiquitin during proliferation and differentiation of neuroblastoma cells. Prostaglandin E1, a stimulator of adenylate cyclase, plus beta-carotene, and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, plus beta-carotene were used to induce terminal differentiation in > 90% of neuroblastoma cells. Changes in ubiquitin level were studied by immunofluorescent staining using either a mouse monoclonal antibody or a rabbit polyvalent antibody to ubiquitin. Results showed that the dividing neuroblastoma cells contained very low levels of ubiquitin localized primarily in the cytoplasm. The intensity of cytoplasmic staining for ubiquitin markedly increased during cAMP-induced differentiation of neuroblastoma cells, being the highest at 4 days after treatment. The neurites of these differentiated cells were also stained, but the nuclei were not. We propose a hypothesis that higher levels of cytoplasmic ubiquitin are needed during cAMP-induced differentiation of neuroblastoma cells for the removal of proteins responsible for cell proliferation through rapid degradation and/or inhibition of transcription, later leading to terminal differentiation.

Characterization and transplantation of two neuronal cell lines with dopaminergic properties.

Immortalized rat mesencephalic cells (1RB3AN27) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP2) in culture. Treatment of 1RB3AN27 and NBP2 cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB3AN27 cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB3AN27 cells exhibited beta- and alpha-adrenoreceptors, and prostaglandin E1 receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D1) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKC alpha and PKC beta isoforms were higher than those of PKC gamma and PKC delta in 1RB3AN27 cells. The 1RB3AN27 cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP2 cells. The grafted 1RB3AN27 were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.

Characterization of human and rat immortalized clones parotid acinar cells with respect to specific proteins and their mRNAs, and receptor-linked adenylate cyclase.

This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP1G). The levels of alpha-amylase mRNAs detected when using alpha-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP1G cells. In contrast to alpha-amylase mRNAs levels, the alpha-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2PH1G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the same alpha-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP1G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP1G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E1 (PGE1) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP1G cells, suggesting that the PGE1 receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.

Establishment of human parotid pleomorphic adenoma cells in culture: morphological and biochemical characterization.

This study reports the establishment of alpha-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP1) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP1 produce low levels of alpha-amylase and relatively high levels of alpha-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP1 are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation of alpha-amylase in these cells.

Establishment and characterization of immortalized cell lines from rat parotid glands.

This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV3neo and pSV5neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV5neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV3neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen, alpha-amylase mRNAs of 1176 and 702 bp, and alpha-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.