Basal Cells Of Stratified Epithelia Research Articles

ERAS, a Member of the Ras Superfamily, Acts as an Oncoprotein in the Mammary Gland

Simple SummaryThe genes of the RAS family are among the group of genes most frequently mutated in human cancer. ERAS is a relatively unknown gene of this family. Although ERAS is overexpressed in some tumoral samples and in several cancer cell lines of human origin, it is not known if its expression drives tumor formation or if, alternatively, its expression is a secondary event in tumoral transformation. In this report, in order to clarify the role of ERAS in mammary tumorigenesis, we studied transgenic mice expressing ERAS in myoepithelial cells of mammary and other exocrine glands and in basal cells of stratified epithelia. These mice displayed an altered development and function of the mammary glands, and suffered high-frequency tumoral lesions in the mammary glands resembling a rare human breast tumor named malignant adenomyoepithelioma. Our results clearly demonstrate that ERAS is a true oncogene able to produce mammary tumors when inappropriately expressed.ERAS is a relatively uncharacterized gene of the Ras superfamily. It is expressed in ES cells and in the first stages of embryonic development; later on, it is silenced in the majority of cell types and tissues. Although there are several reports showing ERAS expression in tumoral cell lines and human tumor samples, it is unknown if ERAS deregulated expression is enough to drive tumor development. In this report, we have generated transgenic mice expressing ERAS in myoepithelial basal cells of the mammary gland and in basal cells of stratified epithelia. In spite of the low level of ERAS expression, these transgenic mice showed phenotypic alterations resembling overgrowth syndromes caused by the activation of the AKT-PI3K pathway. In addition, their mammary glands present developmental and functional disabilities accompanied by morphological and biochemical alterations in the myoepithelial cells. These mice suffer from tumoral transformation in the mammary glands with high incidence. These mammary tumors resemble, both histologically and by the expression of differentiation markers, malignant adenomyoepitheliomas. In sum, our results highlight the importance of ERAS silencing in adult tissues and define a truly oncogenic role for ERAS in mammary gland cells when inappropriately expressed.

Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

P63 in Squamous Cell Carcinoma of the Skin: More Than a Stem Cell/Progenitor Marker

The p63 gene is often overexpressed in squamous cell carcinomas; however, how its overexpression contributes to tumor formation and expansion is still incompletely understood. Devos etal. report the development of a versatile mouse model demonstrating that p63 facilitates squamous cell carcinoma formation in skin and providing an excellent tool to dissect the relevance of its downstream signaling pathways in tumorigenesis.

253 Use of Transfer Factor in Patients with Persistent Genital Human Papillomavirus Infection

BackgroundHuman papillomavirus (HPV) is the most common sexually transmitted infection worldwide. About 75 to 80% of sexually active Americans will be infected with HPV at some point in their lifetime. The risk of HPV infection seems to be related with age at first intercourse, younger age and number of sexual partners. HPV infection is limited to the basal cells of stratified epithelium of the skin or mucous membranas. There is a wide latency period, from months to years, before squamous intraepithelial lesions develop. Most HPV infections are cleared within 2 years by the immune system. Only in 5% to 10% of infected women with “high risk” types the infection persists determining a high risk of developing intraepithelial neoplasias, as cervical cancer, vulvar cancer, penile cancer, and/or anal cancer. The gynecological evaluation and Papanicolau smear are the primary screening tools for detecting HPV infection. There is currently no specific treatment for HPV infection. The Transfer Factor (TF) or Dialyzable Leukocyte Extract is an immunomodulator that has been successfully used as an adjuvant in the treatment of intracelular infections such as recurrent herpes virus diseases. TF induces the expression of RNAm and IFN-γ and increases CD4+ cells. The IFN-γ activates macrophages, neutrophils, B lymphocytes, NK cells, and favours the differentiation of T cells into Th1 lymphocytes that are requiried for the control of intracellular patho gens.MethodsWe used TF in a group of patients with persistent genital human papillomavirus infection.ResultsWe included 12 patients, aged 19 to 45 years old (mean 30), with 14 to 23 years at first intercourse and a mean of 3 sexual partners in their lifetime. All of them had persistent HPV that had been treated before with local and ablative therapeutic options (including cervical freezing, cauterizing loop, imiquimod, podophyllin and/or cervical conization). Transfer factor was administered daily for 5 days, and subsequently at 7-day intervals for 5 weeks. We found an important improvement in the gynecological evaluation of cervix and perineal lesions and a significant reduction in the frequency of relapses.ConclusionsTransfer factor could be used as an adjuvant in patients with persistent genital human papillomavirus infection.

Abstract A52: Overexpression of Sp2 increases susceptibility to wound- and carcinogen-induced papillomas in vivo and inhibits cell cycle progression and induces apoptosis of epidermal stem cells in vitro

Abstract Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct genes (Sp1–9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Inactivation of Sp2 function in zebrafish and mice indicates that Sp2 is required for the completion of gastrulation and embryogenesis, respectively, and thus Sp2 is an essential gene. A causal relationship between Sp2 and tumorigenesis is also suggested by the observation that Sp2 expression is correlated directly with the progression of human prostate cancers and murine cutaneous squamous cell carcinomas. To determine if this correlation is functionally meaningful we created transgenic mice that over-express mouse Sp2 in basal cells of stratified epithelia. We reported recently that these animals (Sp2-C) exhibit increased susceptibility to wound- and carcinogen (DMBA/TPA)-induced tumorigenesis, and we concluded that Sp2 over-expression is oncogenic. To determine whether wound-induced papillomas depend on infiltrating T-cells for growth we treated transgenic animals with Tacrolimus, a well-characterized T-cell inhibitor, before and after surgical wounding. We find that Tacrolimus treatment in Sp2-C, but not wild-type animals, increased wound-induced papillomagenesis dramatically indicating that a T-cell mediated immune response limits the outgrowth of these lesions. To determine if Sp2 DNA-binding activity is required for the increased susceptibility to wound- and carcinogen-induced tumorigenesis, we generated a transgenic mouse line (Sp2-Not1) that over-expresses a mutated Sp2 cDNA lacking a functional DNA binding domain. Interestingly, the incidence of papillomagenesis in Sp2-Not1 mice is half that of wild-type littermates when treated with DMBA/TPA. Moreover, Sp2-Not1 mice do not develop wound-induced neoplasms. Instead Sp2-Not1 mice exhibit a decreased capacity to heal surgical wounds. We interpret these results to indicate that the transgene carried by Sp2-Not1 mice negatively regulates keratinocyte proliferation and/or viability in vivo. To determine the molecular mechanisms underlying the phenotypes presented by our transgenic strains we have prepared primary keratinocyte cultures and analyzed rates of proliferation, expression of differentiation-specific markers, cell-cycle progression, and the incidence of apoptosis. CyQUANT® cell proliferation and tritiated-thymidine incorporation assays showed that keratinocytes obtained from Sp2-C and Sp2-Not1 animals proliferate poorly in vitro compared to wild-type keratinocytes. Flow cytometric analyses indicated that Sp2-C and Sp2-Not1 keratinocytes progress through the cell cycle inefficiently and accumulate in all cell-cycle compartments. Flow cytometry following annexin V/PI staining, immunohistochemistry for cleaved caspases-3, and genomic DNA laddering assays indicate increased numbers of apoptotic cells in Sp2-C and Sp2-Not1 cultures relative to cultures prepared from wild-type littermates. In summary, (1) Sp2 over-expression in epidermal progenitor cells is oncogenic and requires Sp2 DNA-binding activity. (2) Wound-induced papillomagenesis in Sp2 transgenic mice is inhibited by infiltrating T-cells. (3) Sp2 over-expression inhibits progression through all cell-cycle compartments in vitro and induces programmed cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A52.

Induction of ΔNp63 by the Newly Identified Keratinocyte-Specific Transforming Growth Factor β Signaling Pathway with Smad2 and IκB Kinase α in Squamous Cell Carcinoma

The expression of p63 (TP63/p51) occurs in the basal cells of stratified epithelia and is strongly enhanced at the early stages of squamous cell carcinomas (SCCs) of the head and neck, skin, cervix, and others. We analyzed a promoter/enhancer region (2kΔN) that drives the predominant expression of ΔNp63 for sensitivity to Smad signaling pathways. Reporter assays in HepG2 cells showed a moderate activation of 2kΔN by Smad2 and IκB kinase α (IKKα), partners of the newly identified keratinocyte-specific transforming growth factor β (TGF-β) signaling, but not by other Smad molecules. In A431 cells, 2kΔN was activated by Smad2 and IKKα, for which a Smad binding element (SMD2) at -204 was essential. Binding of Smad2 to the chromosomal SMD2 site was detectable. The association of Smad2 with IKKα was evident in the nucleus of A431, accounting for the enhancement of ΔNp63 expression by TGF-β. Moreover, both ΔNp63 and IKKα were necessary to maintain the noninvasive phenotype of this cell line. FaDu, an invasive, Smad4-deficient SCC, also allowed 2kΔN transactivation by transfected Smad2 in the presence of endogenous IKKα. Reflecting the lack of chromosomal SMD2-Smad2 association and the absence of nuclear IKKα, however, endogenous ΔNp63 was not controlled by TGF-β or IKKα in FaDu. SCC tissue arrays showed nuclear accumulation of IKKα and p63 intensification in well-differentiated noninvasive lesions. This study indicates that p63 is a target gene of the proposed keratinocyte-specific TGF-β signal pathway for suppression of the malignant conversion of SCC.

ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, DeltaNp63alpha, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that DeltaNp63alpha functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing DeltaNp63alpha. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of DeltaNp63alpha decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that DeltaNp63alpha maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of DeltaNp63alpha together with p53 inactivation in SCCs.

Abstract C4: Sp2: A regulator of stem cell differentiation and tumorigenesis

Abstract Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct proteins (Sp1-9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Previous data from our lab and others has indicated that Sp2 is over-expressed in human prostate cancer and in murine skin squamous cell carcinoma. To determine if the de-regulated expression of Sp2 is oncogenic, transgenic mice over-expressing mouse Sp2 via the keratin 5 (K5) promoter were generated. This promoter drives expression of linked genes in basal cells of stratified epithelia, such as skin. Two different transgenic lines (Family A and C) were derived and characterized. The transgenes carried by Family A and C mice were localized to chromosomes 6 and 5, respectively. Endogenous genes were not disrupted by transgene integration in Family A, whereas transgene integration in Family C mice occurred within the latrophilin 3 (Lphn3/CIRL3) locus. Homozygous Family A and C animals develop normally and are indistinguishable from wild-type and hemizygous littermates until post-natal day 2. Family C animals invariably perish at this time due to skin deterioration whereas homozygous Family A animals develop skin lesions on post-natal day 4, are runted and developmentally retarded and perish prior to post-natal day 13. Immunohistochemical staining revealed that that an expanded population of K5-negative, K14-positive progenitor cells inundates all epidermal cell layers of homozygous Family A animals. Expression of markers of differentiated basal keratinocytes (e.g., keratin 10, loricrin) is decreased in homozygous Family A animals. The expression level of keratin 6, a marker of epidermal wounding, inflammation, and/or tumorigenesis is increased in Family A animals beginning at post-natal day 4 as is the expression and distribution of CD34, a hair follicle stem cell marker. Hemizygous Family A animals progress to adulthood unremarkably, however 70% exhibit varying degrees of alopecia (hair loss) beginning at 1 month of age. Family C hemizygotes do not develop alopecia, however an expanded pool of progenitor cells renders these animals susceptible to wound-induced neoplasia, and the incidence of papilloma formation rises with an age-dependent increase in Sp2 levels. Sp2 expression in Family A hemizygotes is insufficient to promote wound-induced neoplastic growth, but epidermal differentiation is slowed sufficiently to retard rates of wound healing. These and other results have led us to speculate that Sp2 functions to limit stem cell differentiation and thus its over-expression contributes to the outgrowth of poorly-differentiated, tumorigenic cells. Citation Information: Cancer Res 2009;69(23 Suppl):C4.

The Expression and Diagnostic Utility of p63 in the Female Genital Tract

p63 plays a key role in epithelial development in various organs, being expressed in myoepithelial cells and in basal cells of stratified epithelia. In the female genital tract, p63 is expressed in the basal and parabasal cells of mature cervical, vaginal and vulval squamous epithelium, and also in cervical reserve cells at the transformation zone and in immature metaplastic and atrophic cervical squamous epithelium. In this review, the diagnostic utility of p63 immunohistochemical staining in various pathologic scenarios within the female genital tract is discussed. Cervical microglandular hyperplasia is p63 positive with a characteristic subcolumnar location due to expression in reserve cells. There is increased expression in cervical intraepithelial neoplasia, in accordance with the degree of dysplasia. One of the most useful applications of p63 is in the evaluation of problematic cervical carcinomas; most squamous carcinomas exhibit diffuse nuclear immunoreactivity whereas most adenocarcinomas and neuroendocrine carcinomas are negative or focally positive. In conjunction with neuroendocrine markers, p63 is useful in distinguishing between a squamous carcinoma and a small cell or large cell neuroendocrine carcinoma. In the normal endometrium, a population of p63-positive cells is present which may act as a stem cell population and which is increased in various forms of metaplasia. Placental site nodule and epithelioid trophoblastic tumor (lesions derived from chorionic-type intermediate trophoblast) are usually p63 positive whereas placental site reaction and placental site trophoblastic tumor (lesions derived from implantation site intermediate trophoblast) are usually negative; thus, p63 may be useful in the diagnostic algorithm of trophoblastic lesions. p63 positivity in ovarian epithelial tumors is uncommon and largely restricted to squamous and transitional neoplasms, including benign and borderline Brenner tumor. p63 is also positive in cervical transitional metaplasia, Walthard rests, vulval Paget disease secondary to an underlying urothelial malignancy, tubulosquamous polyp of the vagina, and ectopic prostatic tissue in the cervix.

Immunolocalization of farnesoid X receptor (FXR) in mouse tissues using tissue microarray

Farnesoid X receptor (FXR) is a member of the nuclear receptor family and is known to play important roles in bile acid homeostasis, and lipid and glucose metabolism. In this study, to elucidate the systemic physiological functions of FXR, comprehensive immunohistochemical analysis of cell/subcellular localization of FXR and its heterodimer partner, retinoid X receptor (RXR)-alpha, in adult mice tissues was performed using tissue microarray (TMA)-based immunohistochemistry. FXR immunolabeling was observed in the enterohepatic system--including absorptive epithelium in the intestines, hepatocytes and gall bladder epithelium, several epithelial lineage cells including the basal cells of stratified epithelium in the tongue, esophagus, forestomach--skin, corneal epithelium and ciliary body epithelium in the eye and adrenocortical cells--including glandular cells in the zona reticularis/fasciculata. In these FXP-positive cells, FXR was preferentially localized to the nucleus. RXR-alpha was ubiquitously distributed in the nucleus of most cell types, including FXR-positive cell types in the examined tissues. These data suggest that FXR might have various physiological roles, not only in bile acid homeostasis, and lipid and glucose metabolism, but also in the epithelial cell barrier, visual and urinary function through multiple organ systems.

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Assessment of p63 expression in the salivary gland neoplasms adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and basal cell and canalicular adenomas

Purpose The purpose of this study was to determine the extent of p63 immunoreactivity in the malignant salivary gland neoplasms adenoid cystic carcinoma (ACC) and polymorphous low-grade adenocarcinoma (PLGA) and to compare this to the expression of this marker in the benign salivary gland tumors canalicular adenoma and basal cell adenoma. Few studies on the expression of p63 in head and neck salivary gland tumors have been published to date. P63, a selective immunohistochemical marker of basal/stem cells of stratified epithelium and of myoepithelial cells, is a p53 homologue that plays an essential role in both morphogenesis of epidermis and limb development. P63 immunoreactivity has been demonstrated in squamous cell and urothelial carcinomas. It is generally absent in most nonsquamous cell carcinomas. Study design Formalin-fixed paraffin-embedded sections from 49 salivary gland neoplasms, representing 6 canalicular adenomas, 11 basal cell adenomas, 17 PLGA and 15 ACC accessioned from 1989 to 2002 by the Department of Pathology, Long Island Jewish Medical Center, New Hyde Park, NY, were stained with an anti-p63 monoclonal antibody. Results Nuclear p63 reactivity was uniformly positive in PLGA (17/17, 100%). Positive reactivity was also identified in the majority of cases of ACC (13/15, 87%), primarily in the nonluminal myoepithelial-like cells surrounding luminal cells. Canalicular adenoma did not exhibit any p63 immunoreactivity. All basal cell adenomas of parotid origin stained strongly for p63, with staining localized to the peripheral tumor cells situated adjacent to the connective tissue stroma. None of the basal cell adenomas originating in the upper lip stained with p63. In native adjacent salivary gland tissue, p63 reactivity was identified focally in the nuclei of myoepithelial and basal duct cells. Conclusions P63 is strongly expressed in basal cell adenoma of parotid origin, and in ACC and PLGA. Canalicular adenoma did not demonstrate p63 staining, consistent with this tumor's putative luminal ductal cell differentiation. Our results suggest that the neoplastic cells in PLGA may represent either a population of p63-positive epithelial stem/reserve cells similar to the basal cells of stratified epithelium, or modified myoepithelial cells. Given the staining pattern of the tumors examined, p63 does not appear to be an ideal marker for distinguishing between ACC, PLGA, and basal cell adenoma.

The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro.

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5–K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD ∼2 μM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5–K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

Expression of Cytokeratins and Vimentin in Normal and Neoplastic Tissue from the Bovine Female Reproductive Tract

The distribution of cytokeratins (CKs) and vimentin in the normal genital tract of calves and cows at different stages of the oestrous cycle and in epithelial tumours of the tract was studied immunohistochemically. Few differences in CK and vimentin immunolabelling were detected in relation to age or stage of the oestrous cycle. Coexpression of CKs in simple epithelia and in basal cells of stratified epithelia was detected in the oviduct and endocervix; this coexpression was different from that previously described in women. The demonstration of CKs but not vimentin in the neoplastic cells of a serous superficial ovarian papilloma suggested an origin from the ovarian surface epithelium, while the coexpression of CKs and vimentin in serous papillary and mucinous cystadenomas pointed to a possible origin from the rete ovarii. Studies on three uterine adenocarcinomas and the ovarian metastases from two of these showed an endometrial-CK phenotype. The intermediate filament profile of normal endometrium, conserved in uterine adenocarcinomas and their ovarian metastases, may be useful in discriminating between ovarian metastases from endometrial carcinomas and those originating from primary carcinomas in other organs.

Transgenic mice bearing the polyomavirus large T antigen directed by 2.1 kb of the Keratin 19 promoter develop bronchiolar papillary tumors with progression to lung adenocarcinomas

Keratin 19 is an intermediate filament protein produced by cells of simple epithelia and basal cells of stratified epithelia of different organs. These cell types are associated with important human cancers. We have used the keratin 19 promoter to target the expression of the polyomavirus (Py) large T-antigen, an immortalizing oncogene known to bind to the tumor suppressor retinoblastoma gene product, to epithelial cells. Individuals of the transgenic mouse line K19PyLT-6 developed one or two nodules in one of their lungs. By histology, the nodules were papillary tumors that consisted of nonciliated epithelial cells of the terminal bronchioles. In addition, infiltrates emanating from the nodules were consistent with the development of pulmonary adenocarcinomas. In situ hybridization techniques demonstrated large T-antigen expression in the tumors. Primary cultures were established from a lung tumor dissected from a K19PyLT-6 transgenic mouse. These large T-antigen-expressing cell lines produced the keratin proteins reminiscent of the epithelial origin of the lung tumor. However, further molecular studies indicated that these cell lines did not express Clara cells or pneumocytes markers. s.c. injection of the cell lines into nontransgenic syngeneic mice produced tumors in 2 weeks that resembled malignant pulmonary adenocarcinomas. These animals, which display tumor progression in situ, and the cell lines derived thereof provide a useful system for the study of lung tumorigenesis.

Enhanced extrinsic innervation of nasal and oral chemosensory mucosae in keratin 14-NGF transgenic mice

The role of nerve growth factor (NGF) in neurotrophic support for the extrinsic innervation of the nasal and oral mucosae was investigated in keratin 14 (K14) - NGF transgenic mice in which NGF was overexpressed in K14-synthesizing cells. K14 immunoreactivity was localized in the epithelial basal cells of the whisker pad skin, the hard palate, the floor of the ventral meatus, and the anterior tongue that are stratified squamous epithelia, and also in basal cells of the vomeronasal, olfactory, and respiratory epithelia that are non-stratified epithelia. In transgenic mice, NGF expression was identified and confined primarily to the basal cells of stratified epithelia. The nasal mucosae including the vomeronasal, olfactory, and respiratory mucosae, and the glands associated with the vomeronasal organ received a greater innervation of protein gene product 9.5-immunoreactive extrinsic fibers in transgenic animals than nontransgenic controls. An increased density of calcitonin gene-related peptide-immunoreactive extrinsic fibers was observed in the nonsensory epithelia of the vomeronasal organ, the olfactory sensory and respiratory epithelia in transgenic animals. Our results indicated that the hyperinnervation of the nasal and oral mucosae by extrinsic neurons is due at least partially to target-derived NGF synthesis and release by K14-expressing basal cells.

Cytokeratins as markers of initial stages of squamous metaplasia in feline mammary carcinomas

Summary Expression of keratins (cytokeratins, ck) known to be suitable markers for different types of epithelial differentiation was analyzed in specimens of feline mammary tissue. A panel of specific anti-ck monoclonal antibodies (MAb) was used to determine ck distribution pattern in normal feline tissues (n = 3), and in benign (n = 18) and malignant (n = 20) feline mammary tumors. In selected tumors, the ck distribution pattern was also determined by biochemical methods. A MAb specific for α-smooth muscle actin was used to discriminate between myoepithelial cells and luminal epithelial cells. In normal mammary gland tissues, 6 MAb reacted exclusively, either with myoepithelial cells or with luminal epithelial cells. Luminal epithelial cells reacted with MAb specific for ck typical of simple epithelia, whereas myoepithelial cells reacted with MAb specific for ck in basal cells of stratified epithelia. A similar distribution of ck was detected in specimens from benign tumors, except that ck4 was not detected in normal mammary gland tissues and was detected in some ducts in specimens with adenosis. Almost all tumor cells in specimens from malignant tumors reacted with MAb specific for ck typical of simple epithelia. Concomitant expression of ck typical of stratified epithelia was detected in small or large subpopulations of tumor cells in 70% of carcinomas. Cytokeratins typical of basal cell layers and typical for suprabasal layers of inner stratified epithelia were detected. Cytokeratins typical of stratified epithelia were always found in areas of squamous metaplasia, but also were found in adenocarcinomal cells surrounding these areas. We postulate that these adenocarcinoma cells represent transitional cells where the initial stages of squamous metaplasia have taken place, but squamous metaplasia is not yet detectable by light microscopy.

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin

Abstract We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible “neoexpression” during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues.

Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.

Characterization of a monoclonal antibody (BG3C8) that reacts with basal cells of stratified epithelia.

Monoclonal antibodies were produced against a suspension of formaldehyde fixed human epidermal cells. The supernatant fluid of one clone (BG3C8) yielded a bright immunofluorescent staining of basal cells both in cryostat sections of human split skin and in preparations of purified basal cells. As determined by one- and two-dimensional gel immunoblotting of epidermal basal cell proteins the antibody recognized a minor basic polypeptide of 55,000 apparent molecular weight that was not present in extracts of cultured cell lines of epithelial, fibroblast and lymphoid origin. The distribution of the 55,000 molecular weight protein in normal human tissue was determined by immunohistological staining of cryostat tissue sections that included: central nervous, endocrine, female and male reproductive, alimentary, lymphatic-haemopoietic, respiratory and urinary systems, skin and its appendages, mesenchymal tissue (bone, cartilage, muscle, connective tissue, blood vessels, nerves and synovia) as well as placenta and umbilical cord. The results showed a restricted distribution of this antigen which was found only in basal cells of most stratified or pseudostratified epithelia and in myoepithelial cells. This antibody may be useful in the study of normal and pathological differentiation in various epithelial disorders.