Squamous Morphology Research Articles

New Pathologic Classification of Lung Cancer: Relevance for Clinical Practice and Clinical Trials

We summarize significant changes in pathologic classification of lung cancer resulting from the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) lung adenocarcinoma classification. The classification was developed by an international core panel of experts representing IASLC, ATS, and ERS with oncologists/pulmonologists, pathologists, radiologists, molecular biologists, and thoracic surgeons. Because 70% of patients with lung cancer present with advanced stages, a new approach to small biopsies and cytology with specific terminology and criteria focused on the need for distinguishing squamous cell carcinoma from adenocarcinoma and on molecular testing for EGFR mutations and ALK rearrangement. Tumors previously classified as non-small-cell carcinoma, not otherwise specified, because of the lack of clear squamous or adenocarcinoma morphology should be classified further by using a limited immunohistochemical workup to preserve tissue for molecular testing. The terms "bronchioloalveolar carcinoma" and "mixed subtype adenocarcinoma" have been discontinued. For resected adenocarcinomas, new concepts of adenocarcinoma in situ and minimally invasive adenocarcinoma define patients who, if they undergo complete resection, will have 100% disease-free survival. Invasive adenocarcinomas are now classified by predominant pattern after using comprehensive histologic subtyping with lepidic, acinar, papillary, and solid patterns; micropapillary is added as a new histologic subtype with poor prognosis. Former mucinous bronchioloalveolar carcinomas are now called "invasive mucinous adenocarcinoma." Because the lung cancer field is now rapidly evolving with new advances occurring on a frequent basis, particularly in the molecular arena, this classification provides a much needed standard for pathologic diagnosis not only for patient care but also for clinical trials and TNM classification.

Steroid Signaling within Drosophila Ovarian Epithelial Cells Sex-Specifically Modulates Early Germ Cell Development and Meiotic Entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. Our results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals.

Immunohistochemical profile to distinguish urothelial from squamous differentiation in carcinomas of urothelial tract

Urothelial neoplasms with squamous morphology raise the differential diagnosis between pure primary squamous cell carcinoma, urothelial carcinoma with squamous differentiation and secondary involvement by squamous cell carcinoma, for example, from uterine cervix. Accurate identification between these entities is critical due to differing prognosis and therapeutic strategies. We evaluated the utility of an immunohistochemical panel of 3 urothelial-associated antibodies (uroplakin III, S100P, and GATA3) and two squamous-associated antibodies (CK14 and desmoglein-3) in 50 primary urothelial neoplasms: 15 pure urothelial carcinomas, 12 pure squamous cell carcinomas and 23 urothelial carcinomas with squamous differentiation. Squamous differentiation was defined by intercellular bridges or evidence of keratinization. Pure squamous cell carcinomas were positive for CK14 (100%) and desmoglein-3 (75%), negative for GATA3 and uroplakin III; one case was S100P positive (9%). Pure urothelial carcinomas had an opposite pattern and were positive for S100P (93%), GATA3 (93%), and uroplakin III (67%) and were negative for desmoglein-3; CK 14 was positive in 27% of cases; 74% of urothelial carcinomas with squamous differentiation had expression of urothelial and squamous associated markers (S100P, 83%; GATA3, 35%; uroplakin III, 13%; CK14, 87%; and desmoglein-3, 70%), although reactivity for individual markers within some tumors did not always correspond with morphologic differentiation. Of the remaining 26%, 4 showed an overall "squamous" immunoprofile, whereas 2 cases showed a "urothelial" immunoprofile. Our study showed that a panel of five antibodies identifies squamous and urothelial differentiation in most instances suggesting potential diagnostic utility.

Incidence of colorectal premalignant and malignant neoplasm on colonoscopy in an in-patient setting.

e14185 Background: Colorectal cancer (CRC) continues to be a leading cause of death and the third most common cancer in both genders. Numerous studies have shown that routine screening for colon cancer reduces CRC incidence and mortality by detecting premalignant lesions early. Hence screening colonoscopy in the outpatient setting has been the standard of care. In contrast, the incidence of pre-malignant (PMN) and malignant neoplasms (MN) identified during a colonoscopy due to an acute presentation in an in-patient setting is currently unknown. We evaluate the incidence of PMN and MN lesions identified in patients that underwent in-patient colonoscopy for anemia, gastrointestinal bleed (GIB), and or abdominal pain. Methods: A retrospective review of all gastrointestinal consultations (GIC) from January 1st, 2009 until December 31st, 2009 at a single institution. GIC as determined by the requesting provider for anemia, abdominal pain, and/or gastrointestinal bleed (GIB) were included. Results: There were 291 GICs for anemia, abdominal pain, GIB, or a combination thereof resulting in 218 colonoscopies. A total of 15 (6.9%) PMN and MN lesions were identified. Pathological evaluation reveled 13 (86.7%) of these lesions to be consistent with that of adenocarcinoma. The remaining 2 showed squamous cell cancer morphology. PMN/MNs were identified in patients with GIB and anemia (46.7%), anemia alone (20%), GIB and abdominal pain (20%), and GIB alone (13.3%). There was no significant difference in lesions found between genders (46.7% male). Conclusions: Routine outpatient screening colonoscopy is the standard of care with the goal of resecting early stage cancerous lesions resulting in cure. However, there is paucity of data on the incidence of these lesions being identified in an in-patient setting where colonoscopy is performed for GI bleed, anemia, abdominal pain, or a combination thereof. We demonstrate that colonoscopy resulted in identification of PMN/MNs in a significant proportion of patients with afore mentioned specific complaints. Interestingly, most lesions were identified in patients with GIB and anemia, suggesting that a thorough evaluation be undertaken as it may herald an underlying neoplasm.

Urothelial carcinoma with multiple patterns of divergent differentiation featuring small cell, clear cell, papillary glandular and squamous cell morphology

Urothelial carcinoma with multiple patterns of divergent differentiation featuring small cell, clear cell, papillary glandular and squamous cell morphology

Laparoscopic Anti-Reflux Surgery Improves Daytime Somnolence

Laparoscopic Anti-Reflux Surgery Improves Daytime Somnolence

HIF1α Is Essential for Normal Intrauterine Differentiation of Alveolar Epithelium and Surfactant Production in the Newborn Lung of Mice

Neonatal respiratory distress syndrome (RDS) is mainly the result of perturbation in surfactant production and is a common complication seen in premature infants. Normal fetal lung development and alveolar cell differentiation is regulated by a network of transcription factors. Functional loss of any of these factors will alter the developmental program and impact surfactant production and normal gas exchange. During development, the fetus is exposed to varying oxygen concentrations and must be able to quickly adapt to these changes in order to survive. Hypoxia-inducible factor 1alpha (HIF1alpha) is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension and is essential for normal development. Its role in lung maturation is not well defined and to address this knowledge gap, a lung-specific HIF1alpha knock-out model has been developed. Loss of HIF1alpha early in lung development leads to pups that die within hours of parturition, exhibiting symptoms similar to RDS. Lungs from these pups display impaired alveolar epithelial differentiation and an almost complete loss of surfactant protein expression. Ultrastructural analysis of lungs from HIF1alpha deletion pups had high levels of glycogen, aberrant septal development, and decreased expression of several factors necessary for proper lung development, including HIF2alpha, beta-catenin, and vascular endothelial growth factor. These results suggest that HIF1alpha is essential for proper lung maturation and alteration in its normal signaling during premature delivery might explain the pathophysiology of neonatal RDS.

In Vivo Confocal Microscopic Evaluation of Corneal Wound Healing after Epi-LASIK

To study the healing of corneal wounds after epikeratome laser-assisted in situ keratomileusis (epi-LASIK). Twenty-seven patients who had undergone epi-LASIK for the treatment of myopia or myopic astigmatism in 46 eyes were enrolled. A single intraoperative application of 0.02% mitomycin C (MMC) for 20 to 30 seconds was used in 24 eyes with a refraction of no less than -6.0 D (MMC group). MMC was not given to eyes with myopia less than -6.0 D (non-MMC group). The eyes were examined by in vivo confocal microscopy at 1, 3, and 7 days after surgery and then weekly during the first month and once each at 3 and 6 months. Selected images of the corneal basal-apical surface epithelia and stromal reactions quantified by z-scan profile were evaluated. In vivo confocal microscopy showed that cells in most of the epithelial flaps were damaged during the first few days after surgery and were rapidly replaced by new growing cells. In the MMC and non-MMC groups, the corneal basal epithelial cells returned to their preoperative morphology in 0% and 13.6% of the eyes after 1 week, 37.5% and 36.4% after 2 weeks, and 87.5% and 86.3% after 1 month, respectively. The corneal apical surface epithelial cells in the MMC and non-MMC groups recovered their squamous morphology in 12.5% and 13.6% of the eyes at 2 weeks, 37.5% and 54.5% at 1 month, and 52.4% and 57.9% at 6 months, respectively. There was no difference in the stromal reaction between the groups at 1, 3, and 6 months after surgery. Damage of the epithelial flaps after epi-LASIK was observed by in vivo confocal microscopy. MMC usage may cause more damage to the epithelial flaps. There was no difference in stromal reaction between the groups with and without MMC.

Remarkably High Frequency of EGFR Expression in Breast Carcinomas with Squamous Differentiation

The human epidermal growth factor receptor (EGFR) is reportedly overexpressed in 15-20% of breast carcinomas. EGFR overexpression is associated with reduced survival and is inversely correlated with expression of estrogen receptor (ER). This study assessed EGFR expression in breast carcinomas with squamous differentiation. The immunohistochemical (IHC) expression of EGFR was evaluated in 39 breast carcinomas with squamous differentiation (30 pure squamous, 6 adenosquamous, 3 carcinosarcomas) by use of the pharmDx assay (clone 2-18C9, DakoCytomation). Cases were considered positive if at least 10% of the cells showed 1+ positivity in the squamous component. Squamous differentiation was confirmed with IHC for CK5-6 (clone D5/16B4, DakoCytomation). ER, PR, and HER2 status as well as clinical information regarding treatment and outcome were correlated. As a control, a tissue microarray comprising 280 lymph node positive breast carcinomas was evaluated with the same EGFR assay. The 39 patients ranged in age from 33 to 77 years (mean 52). The tumors measured 1.3-30 cm (mean 4.8). Sentinel or full axillary lymph node dissection was performed in 28 patients. Fourteen patients had positive lymph nodes. At the time of initial diagnosis, 3 patients had distant metastasis. Follow-up was available for 16 patients (mean 45 months). Disease-free survival at 3 years was 70%. Among the 39 tumors 87% (34) were positive for EGFR (p<0.0001). Sixty-nine percent (27 of 39) showed >50% 2+ EGFR staining. EGFR-positive tumor cells (showing squamous morphology) were also found in 1 bone, 1 lung, and 8 of 11 lymph node metastases available for evaluation. All 11 lymph nodes showed squamous differentiation. All but 1 of the EGFR+ tumors examined were ER and PR negative. Six EGFR-positive tumors were HER2 positive. No statistically significant differences in HER2 status, size, lymph node status and disease-free survival were observed between EGFR+ and EGFR- cases, but the number of EGFR-negative tumors was quite small. Nine of 280 (3%) of lymph node-positive invasive carcinomas on the tissue microarray were EGFR+; review of the initial diagnostic slides failed to reveal squamous features in all but 1 of the 9 EGFR+ tumors. Breast carcinomas with squamous differentiation are a distinct subgroup of breast tumors with a very high frequency of EGFR positivity. Breast carcinomas of this type would be ideal candidates for a trial with EGFR inhibitors.

Alcohol dehydrogenase 3 transcription associates with proliferation of human oral keratinocytes.

Gene expression underlying cellular growth and differentiation is only partly understood. This study analyzed transcript levels of the formaldehyde-metabolizing enzyme alcohol dehydrogenase 3 (ADH3) and various growth and differentiation-related genes in human oral keratinocytes. Culture of confluent cells both with and without fetal bovine serum inhibited colony-forming efficiency and induced a squamous morphology. Confluency alone decreased the transcript levels of ADH3, the proliferation markers cell division cycle 2 (CDC2) and proliferating cell nuclear antigen (PCNA), and the basal cell marker cytokeratin 5 (K5), but increased transcripts for the suprabasal differentiation markers involucrin (INV) and small proline-rich protein 1B (SPR1). These changes were variably influenced by serum, i.e., loss of CDC2 and PCNA was inhibited, loss of K5 promoted, increase of SPR1 transcripts inhibited, and increase of INV promoted. The extent and onset of the effects implied that ADH3 transcription serves as a proliferation marker and that confluency with or without serum exposure can serve to selectively analyze proliferative and differentiated cellular states.

Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood–brain barrier model for drug uptake and transport studies

Well-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood–brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Ω cm 2, but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients (×10 −6 cm s −1) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MRP 1 and 5; β-alanine, system L and system y +L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated d-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/4F2hc amino acid transporter was examined through uptake of l-leucine and l-phenylalanine and provided K m and V max values of approximately 16 μM and 350–480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood–brain barrier transport mechanisms.

Genetic Mosaic Analysis Reveals That GATA-4 Is Required for Proper Differentiation of Mouse Gastric Epithelium

During mouse embryogenesis GATA-4 is expressed first in primitive endoderm and then in definitive endoderm derivatives, including glandular stomach and intestine. To explore the role of GATA-4 in specification of definitive gastric endoderm, we generated chimeric mice by introducing Gata4−/− ES cells into ROSA26 morulae or blastocysts. In E14.5 chimeras, Gata4−/− cells were represented in endoderm lining the proximal and distal stomach. These cells expressed early cytodifferentiation markers, including GATA-6 and ApoJ. However, by E18.5, only rare patches of Gata4−/− epithelium were evident in the distal stomach. This heterotypic epithelium had a squamous morphology and did not express markers associated with differentiation of gastric epithelial cell lineages. Sonic Hedgehog, an endoderm-derived signaling molecule normally down-regulated in the distal stomach, was overexpressed in Gata4−/− cells. We conclude that GATA-4-deficient cells have an intrinsic defect in their ability to differentiate. Similarities in the phenotypes of Gata4−/− chimeras and mice with other genetically engineered mutations that affect gut development suggest that GATA-4 may be involved in the gastric epithelial response to members of the TGF-β superfamily.

Cytokeratin expression and epithelial differentiation in Warthin's tumour and its metaplastic (infarcted) variant.

Warthin's tumour is characterized by a bilayered columnar epithelium. Transformation into metaplastic (infarcted) Warthin's tumour includes squamous metaplasia of the epithelium along with regressive changes in the stroma. Misinterpretation of metaplastic Warthin's tumour for malignancy is a serious diagnostic pitfall. This study assesses the utility of cytokeratin expression in Warthin's tumour and its metaplastic variant. Twenty-six cases of Warthin's tumour, among them eight metaplastic Warthin's tumours, were investigated employing immunohistochemistry. Both Warthin's tumour and its metaplastic variant regularly expressed cytokeratins (CK) 7, 8, 18, and 19. Staining results with antibodies to CK10, 10/13, 1/2/10/11, and 20 were negative in all specimens. Immunoreactivity for CK 5/14 and 17 was restricted to basal cells in Warthin's tumour, but involved basal as well as surface cells in metaplastic Warthin's tumour. Warthin's tumour and its metaplastic (infarcted) variant both express CK 7, 8, 18, and 19, which are typical for columnar differentiation. Cytokeratins typical of squamous differentiation are absent from Warthin's tumour and its metaplastic variant, irrespective of the squamous morphology of the epithelium in metaplastic Warthin's tumour. The expression of CK 5/14 and 17, which are typical of regenerative cells, is restricted to basal cells in Warthin's tumour, but is expressed also in surface cells in metaplastic Warthin's tumour.

Cytokeratin 14 as a marker of squamous differentiation in transitional cell carcinomas.

The presence of squamous differentiation in transitional cell carcinomas has been variably related to prognosis and response to radiotherapy. This study sought to establish whether cytokeratin (CK) 14, normally expressed...

Morphology of anal carcinoma: a reappraisal

The morphological heterogeneity of anal carcinomas is well recognised, although the prognostic significance of variable squamous, basaloid and ductal differentiation, by the carcinoma cells is uncertain. The clinical classification is inconsistent due to lack of standard definitions of anal canal and margin. In this study, statistical methods were used to investigate whether discrete groups of tumours can be identified on the basis of shared morphological characteristics. The findings were correlated with the tumour site and prognosis. Principal component analysis on 100 resected cases of anal carcinoma showed that three sets of features were co-expressed by tumour cells: duct formation and diastase Periodic Acid Schiff (dPAS) positivity; keratinization, stratification and the presence of intercellular bridge formation (prickles); basaloid cells, a palisade arrangement, retraction artefact and eosinophilic necrosis. 98% of tumours showed keratinization and two thirds showed at least one basaloid feature. No tumours showed a full set of basaloid characteristics without squamous features. Ductal differentiation (16% tumours) showed a significant association with dPAS positivity and the combination was associated with poor prognosis. A correlation was found between clinical and histological assessment of site, with anal margin carcinoma more likely to show pure squamous morphology than canal tumours. Anal carcinoma should therefore be regarded as keratinizing squamous cell carcinoma. The existence of basaloid carcinoma as a separate entity is not supported by these results. Basaloid and ductal changes represent further pathways of differentiation superimposed on an underlying keratinizing squamous carcinomatous pedigree in these tumours.

Immunohistochemistry of a human type I pneumocyte‐associated protein in lung

A mouse monoclonal antibody to a human lung lavage protein was raised using proteins, with the potential ability to bind surfactant, as the immunogen. The proteins were isolated from cadaver lung lavage. The antibody was tested for its reactivity with lung and other organs. It reacted with type I pneumocytes and some of the nonciliated cells in the surface epithelium of distal bronchioles. Staining was also seen in the cells surrounding the glandular structures, superficial keratinocytes of the skin, endothelium, and nerve sheath cells. With the exception of bronchiolar cells, the stained cells have a squamous morphology, and this protein may serve as a marker or determinant of this characteristic of cells. In pathologic lungs some of the cells in air spaces with "bronchiolarization" of the epithelium exhibited staining for the protein. It could not be ascertained whether the stained cuboidal cells were reactive type II pneumocytes or distal bronchiolar cells. The intraalveolar material in pulmonary alveolar proteinosis did not show remarkable staining for the protein. Even though the protein is not unique to type I pneumocytes, it may serve as a marker for these cells in the study of their development and biology.

Induction of fibronectin gene expression by transforming growth factor beta-1 is attenuated in bronchial epithelial cells by ADP-ribosyltransferase inhibitors.

Transforming growth factor-beta (TGF-beta) exerts several effects on cultured airway epithelial cells including inhibition of proliferation and stimulation of fibronectin gene expression. ADP-ribosylation is one potential regulatory mechanism of gene expression by TGF-beta. We tested this possibility by exposing cultured bovine bronchial epithelial cells to the chemical inhibitor of ADP-ribosyl transferase enzymes, 3-aminobenzamide (3-AB) and, for comparison, 3-aminobenzoic acid (3-ABA), which is structurally similar to 3-AB but which does not inhibit ADP-ribosyl transferases. Exponential cell growth rate (1.2 doublings/day) or cellular morphology observed by phase contrast microscopy were not affected by 3 mM 3-AB or 3-ABA. Neither compound antagonized inhibition of cell division or induction of squamous morphology by TGF-beta 1. In contrast, the sixfold stimulation of fibronectin production by exposure of cells to 30 pM TGF-beta 1 for 48 h was reduced by 50% in the presence of 3 mM 3-AB, whereas 3 mM 3-ABA had no effect. The antagonistic effect was augmented by administration of 3-AB 24 h prior to induction by TGF-beta 1. Northern blot hybridization analyses demonstrated that 3-AB, but not 3-ABA, attenuated the induction of fibronectin mRNA by TGF-beta 1 by up to 50%. These observations may implicate a role of cellular ADP-ribosylation in the regulation of some gene expression by TGF-beta.

Partial suppression of tumorigenicity in a human lung cancer cell line transfected with Krev-1.

A human non-small-cell lung carcinoma cell line, Calu-6 (from an anaplastic carcinoma), was transfected with the Ki-ras-related anti-oncogene Krev-1. Several transfectant lines were obtained that showed a reduced tumorigenicity in nude mice with respect to the parental and control transfected cell lines. This decrease was approximately 50% in tumor incidence at 4 wk after subcutaneous inoculation of the transfected cells. In addition, the volume of the Calu-6 revertant-derived tumors was three to 10 times smaller than that of the equivalent tumors produced by inoculation of the control cell line transfected with the neomycin-resistance gene. Krev-1--transfected cells that exhibited reduced tumorigenicity expressed Krev-1 mRNA and had variable numbers of copies of the Krev-1 gene. Moreover, Krev-1--transfected cells exhibited a more differentiated squamous epithelial morphology than the parental and control cell lines did. Moderately elevated levels of protein kinase C activity were detected in some revertant clones. Such activity correlated with the level of expression of Krev-1 mRNA in most cases. In summary, Krev-1 induced important morphological and biological changes in transfected Calu-6 cells that we interpreted as partial reversion of the malignant phenotype.

Serum-free culture of fractionated bovine bronchial epithelial cells

Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta 1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1 microM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.

Differential regulation of myelin gene expression in SV40 T antigen-transfected rat glioma C6 cells

Rat glioma C6 cells were stably transfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T cells) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected cells was reduced by approximately 40% as compared to control C6 cells. The transfection with T-antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-specific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin-associated glycoprotein (MAG) by 153%. The activity of 2':3' cyclic AMP phosphodiesterase (CNP) was increased by approximately 80% in the C6T cells as compared to untransfected, control cells. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differently regulated implying the existence of several independent regulatory mechanisms.