Squamous Cell Markers Research Articles

Lymphoepithelioma is a nonkeratinizing squamous cell carcinoma with Epstein-Barr virus infection in China.

The objective of this study is to investigate the histogenesis of lymphoepithelial carcinoma (LEC) and its relationship with Epstein-Barr virus (EBV). The expression of EBV was detected using in situ hybridization, and the CK5/6, p63, and p40 expression levels were detected using immunohistochemistry in 45 paraffin-embedded tissues from LEC. In 45 paraffin-embedded LEC tissues from 10 different samples, the positive CK5/6 signals were located in the cell membrane. The positive signals for p63 and p40 were located in the cell nucleus. In all LEC cases, the positive rates of CK5/6, p63, and p40 were 93.3% (42/45), 95.6% (43/45), and 93.3% (42/45), respectively. The positive EBV-encoded RNA (EBER) signals were located in the cell nucleus. In the 45 LEC cases, the expression of EBER was strongly positive with a positive rate of 100% (45/45). LEC is closely related to EBV, and EBV plays an important role in the development of LEC. LEC showed positive squamous cell markers, indicating that the samples contain squamous cell carcinoma (SQCC). LEC is EBV (+) with nonkeratinizing SQCC, and this name better reflects the nature of this disease.

Brush biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine.

Autologous oral mucosal epithelial cell sheets have been used for treating epithelial defects such as cornea and esophagus. The cell source of patients' oral mucosal epithelial cell sheet should be examined in normality because it has individual difference. In this study, oral mucosal epithelial cells were less invasively collected by brush biopsy from the buccal, gingival, labial, and palate mucosa of four healthy volunteer donors without anesthesia, and analyzed the keratin expressions by western blotting and the obtained results were compared with those by immunohistochemistry of each of the native tissues. All of the oral mucosal epithelial cells expressed keratin 4 (K4) and K13, which were mucosal stratified squamous epithelial cell markers. K1 and K10, keratinized epithelial cell markers, were also detected in keratinized tissues such as gingival and palate mucosa. The markers of epithelial basal cells such as p63 and K15 were not detected by brush biopsy-western blotting. Although this method does not include basal layers of oral mucosa, protein expressions of upper layer of lesion area are different from normal. Therefore, brush biopsy-western blotting was extremely less invasive and would contribute to quality control of the fabrication of autologous oral mucosal epithelial cell sheets.

NSCLC Driven by DDR2 Mutation Is Sensitive to Dasatinib and JQ1 Combination Therapy.

Genetically engineered mouse models of lung cancer have demonstrated an important role in understanding the function of novel lung cancer oncogenes and tumor-suppressor genes identified in genomic studies of human lung cancer. Furthermore, these models are important platforms for preclinical therapeutic studies. Here, we generated a mouse model of lung adenocarcinoma driven by mutation of the discoidin domain receptor 2 (DDR2) gene combined with loss of TP53. DDR2(L63V);TP53(L/L) mice developed poorly differentiated lung adenocarcinomas in all transgenic animals analyzed with a latency of 40 to 50 weeks and a median survival of 67.5 weeks. Mice expressing wild-type DDR2 with combined TP53 loss did not form lung cancers. DDR2(L63V);TP53(L/L) tumors displayed robust expression of DDR2 and immunohistochemical markers of lung adenocarcinoma comparable with previously generated models, though also displayed concomitant expression of the squamous cell markers p63 and SOX2. Tumor-derived cell lines were not solely DDR2 dependent and displayed upregulation of and partial dependence on MYCN. Combined treatment with the multitargeted DDR2 inhibitor dasatinib and BET inhibitor JQ1 inhibited tumor growth in vitro and in vivo. Together, these results suggest that DDR2 mutation can drive lung cancer initiation in vivo and provide a novel mouse model for lung cancer therapeutics studies.

Proprotein convertase 5/6A is associated with bone morphogenetic protein‐2‐induced squamous cell differentiation

Results Using a retinoic acid deficiency-induced squamous metaplasia model of HNECs, we observed a significant increase in the expression of PC5/6A, a PC member, and BMP-2, a candidate substrate for PC5/6A. Specific lentiviral shRNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Dec-RVKR-CMK, a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signalling pathways. Dec-RVKRCMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps.

Cytomorphology of sebaceous carcinoma with analysis of p40 antibody expression

Sebaceous carcinomas (SBCs) are aggressive tumors with the potential to cause great morbidity and mortality. Poorly-differentiated tumors may at times pose challenges for the correct diagnosis. p40, a new antibody that targets a short isoform of p63 has been shown as a promising squamous cell marker. In this study, we sought to evaluate cytomorphological features of SBC and p40 expression analysis. A total of 29 previously diagnosed cases of SBCs including fine-needle aspirates and histopathology specimens from various sites were reviewed and studied for p40 expression. p40 nuclear expression was semi-quantitatively assessed. Adequate positive and negative controls of non-small cell lung carcinoma were taken for comparison. Expression pattern of normal sebaceous glands was also analyzed. Of the 29 cases, 13 (44.8%) were from the periocular region. The most common extraocular site was parotid gland. Morphologically tumors were categorized into well- and poorly-differentiated varieties based on extent of sebaceous differentiation. p40 positivity was seen in all cases of cytology aspirates and histology sections with similar intensity. No difference in percentage positivity of cells was recorded in well- and poorly-differentiated tumors. p40 can be a valuable marker when evaluating tumors with possible sebaceous differentiation. Although p40 expression in SBCs is not as useful for the differential diagnosis that includes poorly-differentiated squamous cell carcinoma, this study, for the first time in the literature, highlights an important observation that p40 can be utilized as a marker for sebaceous lineage.

Poorly Differentiated Squamous Cell Carcinoma in an Atomic Bomb Survivor.

Dear Editor: Many epidemiological studies of atomic bomb survivors in Japan demonstrate that exposure to radiation increases the risk of cancers, especially leukemia, in most organs1. The risk of non-melanoma skin carcinoma in atomic bomb survivors is elevated2, and the most common primary skin tumors among 152 skin tumors among patients exposed to atomic bomb radiation were basal cell carcinoma (42%), followed by squamous cell carcinoma (SCC) (26%), Bowen's disease (13%), and malignant melanoma (6%)3. Here, we report a case of poorly differentiated SCC in an atomic bomb survivor. An 86-year-old Japanese woman visited our hospital with a one-month history of subcutaneous tumor in the inguinal region. She noticed a tumor on her the left side of waist when she was 46 years old. She was exposed to atomic bomb radiation in Hiroshima at the age of 19 years. Physical examination revealed a 32×40-mm reddish dome-shaped tumor on her the left side of the waist (Fig. 1A) and a 65×72-mm painful subcutaneous tumor on her left inguinal region (Fig. 1B). Blood examination revealed no abnormal changes. Whole-body computed tomography revealed no remarkable changes. Fluorodeoxyglucose-positron emission tomography revealed abnormal uptake in the tumor of on the waist and left inguinal lymph nodes. Histopathological examination of the waist tumor demonstrated numerous tumor cells throughout the dermis and subcutis (Fig. 1C). Atypical large tumor cells had irregular nuclear contours and prominent nucleoli (Fig. 1D). Immunohistochemistry showed the tumor cells were positive for keratin, CK7, p63, p40, and NSE but negative for CK20, chromogranin A, synaptophysin, thyroid transcription factor-1 (TTF-1), CD56, Melan-A, S-100, and EBER (Fig. 2A~E). The results of the histological and immunohistochemical examinations of the inguinal tumor were same. On the basis of these findings, she was diagnosed with poorly differentiated SCC in the waist and metastasis in the inguinal lymph nodes. Tumors in the waist and inguinal regions were resected, and she was subsequently treated with electron beam radiation (total dose: 59 Gy). Eight months later, a metastatic lesion in the left lung was observed, but she refused additional treatment. Fig. 1 (A) Reddish dome-shaped tumor on the left side of the waist. (B) Painful subcutaneous tumor on the left inguinal region. (C, D) Histopathological examination of the waist tumor (HE C: ×40, D: ×1,000). (C) Numerous tumor cells ... Fig. 2 Immunohistochemical examination of the waist tumor. Tumor cells stained positive for (A) keratin and (B) CK7, and negative for (C) chromogranin A, (D) CK20, and (E) thyroid transcription factor-1 (A~E: ×400). Tumor cells were positive for keratin as well as squamous cell markers p63 and p40. The common markers of neuroendocrine carcinomas, including Merkel cell carcinoma, CK20, chromogranin A and synaptophysin, were negative. TTF-1, a marker of primary thyroid and lung carcinoma, was negative. These immunohistochemical results and the low differentiation pattern of tumors on hematoxylin & eosin staining lead to a final diagnosis of poorly differentiated SCC. Regarding the relationship between the extent of tumor differentiation and atomic bomb radiation exposure, the frequency of poorly differentiated adenocarcinoma of stomach in atomic bomb survivors is significantly higher than that in non-exposed individuals4. Nevertheless, the association between atomic bomb radiation exposure and poorly differentiated SCC in our patient is speculative. A recent study reports multiple basal cell carcinomas were developed in atomic bomb survivors more than 40 years after radiation exposure5. Thus, exposure to atomic bomb radiation might influence the risk of cancers; therefore, dermatologists should perform long-term surveillance for skin carcinomas in atomic bomb survivors.

Proprotein convertase 5/6a is associated with bone morphogenetic protein-2-induced squamous cell differentiation.

Squamous metaplasia in airway epithelium is a pathological process arising from abnormal remodeling/repair responses to injury. Proteolytic maturation of many growth and differentiation factors involved in tissue remodeling is controlled by proprotein convertases (PCs). However, the role of these convertases in airway remodeling remains poorly understood. Using a retinoic acid deficiency-induced squamous metaplasia model of cultured human nasal epithelial cells (HNECs), we observed a significant increase in the expression of PC5/6A, a PC member, and bone morphogenetic protein-2 (BMP-2), a candidate substrate for PC5/6A. Specific lentiviral short hairpin RNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signaling pathways. Dec-RVKR-CMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps. Taken together, PC5/6A is involved in squamous differentiation of HNECs, possibly through up-regulation of the BMP-2/pSmad1/5/8/p38 signaling pathway, pointing to a potential therapeutic target for the prevention of chronic airway diseases that exhibit squamous metaplasia.

Su1963 Salvage ER for Local Failure of Esophageal Cancer After CRT

G A A b st ra ct s the tumors are positive for squamous cell markers, columnar cell markers, and SCJ specific markers such as KRT7 and MUC4. Evaluation of putative cancer stem cell markers shows these tumors are positive for SOX2, SOX9, CD44, but negative for DCAMKL-1, Lrig-1, and Lgr5. In addition, cytokine expression such as TNFa, IL-1b, and IL-11 was significantly elevated in the tumors compared to non-transformed forestomach and grandular stomach. When cultured in matrigel with R-spondin and Wnt1, mJT1 and mJT4 cells grow into organoid like cystic structure with typical crypt appearance. IHC for normal mice shows small area in SCJ positive for KRT19, CD44, and SOX9. Lineage tracing of K19YFP mice shows TAM injection induces YFP positive cells at columnar cells of SCJ. Conclusion; SCJ of mouse stomach contains cells which grow into squamous cell carcinoma and adenocarcinoma in genetically modified condition. These cells may represent a stem cell population of Barrett esophagus as well as SCJ cancer.

P40 (ΔNp63), a Lung Squamous Cell Marker, Can Also Be Used to Label Breast Myoepithelial Cells

Monoclonal p63 (4A4) antibody is conventionally used to mark lung squamous cell carcinoma. However, it also weakly stains the nuclei of up to 30% of lung adenocarcinomas. Another antibody, p40, recognizes ΔNp63, which is a p63 isoform. In a 2012 study,1 p40 was shown to be much more specific than p63, and recent expert advisories suggest that it could replace p63 as a marker of lung squamous cell carcinoma.2Cost control has become a major factor in contemporary practice, and reducing inventory of redundant antibodies, while increasing the number of uses of antibodies remaining on inventory, is an important way of controlling costs. In addition to lung squamous cell carcinoma, p63 is also commonly used to label breast myoepithelial cells. Our community hospital laboratory, which is under a supply and service contract with a company that does not manufacture p63, has been purchasing it from Biocare Medical (Walnut Creek, California), which is 1 of 2 companies that can market p63 in the United States (Ventana Medical Systems, Tucson, Arizona, is the other one). Biocare Medical also holds exclusive license for marketing p40 in the United States. Given our concerns about costs, and the fact that p40 is an antibody to a p63 isoform known to label breast myoepithelial cells,3 we wondered whether monoclonal p40 could replace p63 entirely for these 2 purposes.We tested 10 cases of excision biopsy for breast carcinoma, selecting blocks that contained invasive carcinoma, in situ carcinoma, and proliferative fibrocystic change, and stained them with both monoclonal p63 (4A4) and monoclonal p40 (ΔNp63), at similar dilutions. We reviewed cases blind to knowledge of antibody. The results demonstrated no difference in the staining pattern of the antibodies, in that they both specifically highlighted the nuclei of myoepithelial cells. Because this is far from a definitive explication of relative sensitivities and specificities of the 2 antibodies, we retain other myoepithelial cell–highlighting antibodies such as CD10, which we use as part of a panel.4 We note that the original investigations1 used polyclonal rather than monoclonal p40. We do not anticipate results being substantially different with the polyclonal antibody.The findings of this limited study indicate that monoclonal p40 may be used instead of p63 for the purposes of highlighting breast myoepithelial cells in addition to assisting in the diagnosis of lung squamous cell carcinoma. Community hospital laboratories like ours could benefit from eliminating an antibody from their inventory, while retaining the ability to make accurate diagnoses.The authors have no relevant financial interest in the products or companies described in this article.

IL-4 induces columnar-like differentiation of esophageal squamous epithelium through JAK/PI3K pathway: possible role in pathogenesis of Barrett's esophagus

Barrett's esophagus is characterized by a distinct Th2-predominant cytokine profile (IL-4) from in vivo or ex vivo evidence. The detailed role of cytokines in Barrett's esophagus, particularly whether Th2 cytokines are causative factors driving metaplastic processes, remains unknown. In this study, air-liquid interface-cultured human esophageal epithelial cells were stimulated by a Th2 cytokine, IL-4, and Th1 cytokines, TNF-α and IL-1β, continuously for 10 days. Barrier function was determined by transepithelial electrical resistance. Morphological changes were investigated by hematoxylin and eosin staining. Keratin profile (keratin 7, 8, 13, and 14) and squamous differentiation markers (involucrin) were investigated by RT-quantitative PCR, Western blotting, and immunohistochemical staining. Pharmacological inhibitors were used to identify the underlying cellular signaling. We report that IL-4, TNF-α, and IL-1β decrease barrier function, but only IL-4 significantly increases cell layers and changes cell morphology. IL-4 time dependently downregulates the expression levels of the squamous cell markers involucrin and keratin 13 and upregulates the expression levels of the columnar cell markers keratin 7 and 8. Neither TNF-α nor IL-1β shows any effect on these indexes. JAK inhibitor I and PI3K inhibitors significantly block the IL-4-induced changes in the levels of keratin 8 and 13. In conclusion, IL-4 inhibits squamous differentiation program of esophageal epithelial cells and induces differentiation toward columnar cells through the JAK/PI3K pathway. Thus IL-4 may be involved in the early stages of Barrett's esophagus development.

Large cell carcinoma of the lung: clinically oriented classification integrating immunohistochemistry and molecular biology

This study aimed at challenging pulmonary large cell carcinoma (LLC) as tumor entity and defining different subgroups according to immunohistochemical and molecular features. Expression of markers specific for glandular (TTF-1, napsin A, cytokeratin 7), squamous cell (p40, p63, cytokeratins 5/6, desmocollin-3), and neuroendocrine (chromogranin, synaptophysin, CD56) differentiation was studied in 121 LCC across their entire histological spectrum also using direct sequencing for epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and FISH analysis for ALK gene translocation. Survival was not investigated. All 47 large cell neuroendocrine carcinomas demonstrated a true neuroendocrine cell lineage, whereas all 24 basaloid and both 2 lymphoepithelioma-like carcinomas showed squamous cell markers. Eighteen out of 22 clear cell carcinomas had glandular differentiation, with KRAS mutations being present in 39 % of cases, whereas squamous cell differentiation was present in four cases. Eighteen out of 20 large cell carcinomas, not otherwise specified, had glandular differentiation upon immunohistochemistry, with an exon 21 L858R EGFR mutation in one (5 %) tumor, an exon 2 KRAS mutation in eight (40 %) tumors, and an ALK translocation in one (5 %) tumor, whereas two tumors positive for CK7 and CK5/6 and negative for all other markers were considered adenocarcinoma. All six LCC of rhabdoid type expressed TTF-1 and/or CK7, three of which also harbored KRAS mutations. When positive and negative immunohistochemical staining for these markers was combined, three subsets of LCC emerged exhibiting glandular, squamous, and neuroendocrine differentiation. Molecular alterations were restricted to tumors classified as adenocarcinoma. Stratifying LCC into specific categories using immunohistochemistry and molecular analysis may significantly impact on the choice of therapy.

Co-opted JNK/SAPK Signaling in Wnt/β-catenin–Induced Tumorigenesis

Aberrant stimulation of the canonical Wnt pathway induces mammary tumorigenesis in mice. It has been well documented that two types of tumors, adenocarcinoma and adenocarcinoma with squamous metaplasia, develop in these mutants. However, the molecular mechanism underlying the induction of squamous transdifferentiation remains largely unknown. Here, we show that JNK/SAPK signaling plays an important role in Wnt-dependent mammary development and malignant transformation. The JNK/SAPK pathway is stimulated in pregnancy-mediated lobulo-alveolar morphogenesis, a process highly dependent on Wnt/β-catenin signaling. Strong elevations of JNK/SAPK signaling are associated with squamous metaplasia of the Wnt-induced adenocarcinoma. Reconstitution of β-catenin and JNK/SAPK signaling activities also promotes expression of the squamous cell marker in cultured epithelial cells. Furthermore, a synergistic activation of these two pathways can be identified in the malignant squamous cells of human endometrial and lung cancers. This is potentially a significant discovery in modern cancer therapy because of the effectiveness of an angiogenesis inhibitor, Avastin, for the treatment of adenocarcinoma, but not squamous cell carcinoma, in human lung cancers. Our finding may improve the usage of biomarkers to distinguish these two poorly differentiated tumor types, sharing similar histologic features.

Isolation and Characterization of Cultured Human Conjunctival Goblet Cells

To isolate and characterize goblet cells from normal human conjunctival tissue to determine whether epidermal growth factor (EGF) receptors are present and whether EGF can influence goblet cell proliferation. Goblet cells were isolated from explant cultures established from normal conjunctival tissue harvested from patients during periocular surgery. The cells were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy, molecular biology, and biochemistry. Proliferation was determined with a MTT proliferation assay after exposing goblet cells, which had been serum deprived for 48 hours, to increasing concentrations of epidermal growth factor (EGF; 0-80 ng/mL) for 24 hours. Goblet cells were isolated from conjunctival explants by scraping nongoblet cells from the culture dish. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff (PAS) reagent, goblet cell-specific cytokeratin-7, HPA lectin, and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-4. The mRNA for MUC5AC was detected using RT-PCR. The presence of the EGF receptors EGFR, ErbB2, and ErbB3 was confirmed through Western blot analysis of cell lysates. EGF elicited a concentration-dependent increase in goblet cell proliferation of 160% +/- 0.5%, 188% +/- 0.45%, 293% +/- 1.3%, and 220% +/- 0.5% of control values with 10, 20, 40, and 80 ng/mL EGF, respectively. Human goblet cells that retain characteristics of goblet cells in vivo can be cultured. EGF receptors are present in human goblet cells, and EGF stimulates their proliferation.

Expression of a squamous cell marker, the spr1 gene, is posttranscriptionally down-regulated by retinol in airway epithelium.

Vitamin A (retinol) is required for the normal mucociliary differentiation of respiratory epithelium. A depletion of vitamin A promotes squamous cell metaplasia. To understand how vitamin A suppresses squamous cell differentiation, the expression of a squamous cell differentiation marker, the small proline-rich protein gene (spr1), was studied in cultured monkey tracheobronchial epithelial (TBE) cells. The expression of the spr1 gene was inhibited about 40 fold by retinol. The mRNA levels of the spr1 gene started to decline within 6 h of retinol treatment and reached a minimum level after 7 days. The inhibition by retinol was concentration dependent and did not require concurrent protein synthesis. The inhibition of the spr1 mRNA by retinol was not due to a decrease in the transcription rate of its gene but due to a decrease in its stability, as determined by nuclear run-on assays and mRNA half-life measurement, respectively. This result was further supported by a DNA transfection study using a chimeric construct containing the spr1 promoter region and the chloramphenicol acetyltransferase (CAT) reporter gene. The CAT activity in transfected cells was not inhibited by retinol. These results suggest that spr1 gene expression is posttranscriptionally down-regulated by retinol.

An unusual expression of a squamous cell marker, small proline-rich protein gene, in tracheobronchial epithelium: differential regulation and gene mapping.

An unusual expression of a putative squamous cell marker, small proline-rich protein (spr1), in mucociliary epithelial cells of conducting airways was demonstrated in a serum-free culture system. A cDNA clone was isolated from the cDNA library of monkey tracheobronchial epithelial (TBE) cells by differential hybridization. This cDNA clone, MT5, exhibited 98% homology to a DNA sequence obtained from human keratinocytes treated with either UV light or phorbol esters (T. Kartasova et al., 1988, Mol. Cell. Biol. 8:2195-2230). The predicted peptide of MT5 is unusual for its high content of proline (29%), glutamine (18%), and cysteine (9%) and its repeated PKVPEPC units. The level of spr1 mRNA in cultured cells was inhibited more than 90% by vitamin A. In contrast, phorbol 12-myristate 13-acetate (PMA) stimulated the level of spr1 mRNA by 3- to 8-fold. This differential regulation coincided with the effects of these chemicals on the cornification of cultured TBE cells. Using MT5 as a probe, we have localized the tracheal spr1 gene on the human chromosome 1 by a Southern blot analysis using a panel of human-rodent somatic cell hybrid DNAs. The gene was further sublocalized to bands q22-23 by in situ hybridization.

Retinoic acid receptors as regulators of human epidermal keratinocyte differentiation.

To examine the role of nuclear retinoic acid (RA) receptors (RARs) in the regulation of squamous differentiation in normal human epidermal keratinocytes (NHEK), we analyzed binding activity, mRNA expression, and transcriptional activity of the endogenously expressed RARs. Specific RA-binding activity eluted from size-exclusion HPLC with an apparent mol wt of 50 kilodaltons and was predominantly (greater than 95%) associated with the NHEK nuclear cell fraction. This RAR-binding activity represented in part the expression of RAR alpha and RAR gamma genes, whose transcripts were expressed in similar abundance in undifferentiated NHEK. Differentiation resulted in lower mRNA expression of RAR alpha relative to the mRNA expression of RAR gamma. Treatment of NHEK cells with 10(-6) M RA did not induce expression of RAR beta mRNA. Similarly, three squamous cell carcinoma cell lines derived from human skin and oral cavity expressed RAR alpha and RAR gamma transcripts, but not RAR beta transcripts. Transfection of NHEK with chloramphenicol acetyltransferase (CAT) reporter plasmids indicated that the endogenously expressed RARs could activate transcription through the RAR beta response element in a concentration-dependent manner with doses of 10(-9) M RA and higher. CAT expression was not activated through TRE, a palindromic thyroid hormone response element with purported RA responsiveness. The competitive binding of benzoic acid derivatives of RA to RAR correlated with the ability of each analog to suppress mRNA expression of the squamous cell markers, involucrin, type I transglutaminase, and SQ37, and to activate transcription of the RAR beta response element-CAT reporter. These results demonstrate that the control of NHEK differentiation by RA is consistent with the interaction of the retinoid with RAR and the regulation of transcription by that ligand-receptor complex.

Calcium Regulation of Antigen Expression on Normal and Malignant Human Squamous Cells In Vitro

In vitro, normal keratinocytes exhibit undifferentiated morphologic features and proliferate for multiple passages in low-calcium medium (less than or equal to 0.3 mmol/L) whereas, in high-calcium medium (greater than or equal to 1.0 mmol/L), these cells assume differentiation characteristics, begin to stratify, and eventually cease proliferating. In contrast, malignant keratinocytes grow well in high-calcium medium. Expression of pemphigus vulgaris antigen, a squamous cell marker, is altered on cultured normal keratinocytes by calcium. In this study we compared the effects of calcium levels on expression of cell surface antigens by UM-SCC-38, a human squamous carcinoma cell line, and normal keratinocytes cultured from newborn foreskin. Pemphigus, pemphigoid, beta 2-microglobulin antigens, as well as the epidermal growth factor receptor and the A9 germinal epithelial cell basement membrane squamous carcinoma antigen were examined. Pemphigus antigen was strongly expressed on normal and malignant cells in high-calcium but not low-calcium medium. Calcium concentration did not affect the expression of any of the other antigens tested. Thus, although calcium induces differentiation and eventual loss of proliferative capacity in normal but not malignant keratinocytes in vitro, we were unable to demonstrate differences in pemphigus vulgaris antigen expression that might be linked to the growth inhibitory effects induced by high calcium levels in nontransformed epithelial cells in culture.

Transient Left Bundle Branch Block Following Exertion

Morular metaplasia (MM) is a peculiar type of metaplastic change commonly observed in endometrial lesions, which is defined by the absence of overt squamous features and a characteristic immunophenotype. The nature of MM and its relationship with conventional squamous differentiation (SD) is still undefined.Here, we present a morphological and immunophenotypical study of cases with mixed MM/SD and conventional SD, providing new insights on this field.Twenty cases of endometrioid carcinoma (10 with mixed MM and SD and 10 with conventional SD) were assessed by immunohistochemistry for β-catenin, CD10, CDX2, ki67, p63, p40, estrogen receptor (ER), progesterone receptor (PR) and cytokeratins (CK) 5/6, 7, 8/18 and 19.In mixed MM/SD cases, SD was mostly located within the MM areas; several degrees of SD development were observed within MM, from cells with larger cytoplasm and prominent membrane, to overt SD with morular shape and ghost cell keratinization. In the MM→SD transition, there was progressive loss of nuclear β-catenin, CD10, CDX2 and CK8/18 expression, increase of CK5/6 and CK7 expression, and stable CK19 positivity. ER, PR and ki67/MIB1 expression was low-to-negative in both MM and SD. The squamous cell markers p63 and p40 were mostly expressed at the interfaces between MM and SD. Conventional SD cases showed direct transition from glandular epithelium to SD with a surface growth and no ghost cell keratinization; immunohistochemistry showed strong positivity for ER, PR and all CKs, basal positivity for p63, p40 and ki67/MIB1, negativity for nuclear β-catenin, CD10 and CDX2.In conclusion, MM appears as the precursor of a peculiar form of SD, which differs morphologically and immunophenotypically from conventional SD. Defining MM based on the absence of overt squamous might not be meaningful. Further studies are necessary to clarify the nature of MM.