Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic culture.

The patterns of expression of keratins K1 and K8, filaggrin, and the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), were studied in normal and human papillomavirus (HPV) 16 or 18 immortalized keratinocyte cell lines grown on organotypic raft cultures. Normal keratinocytes produced an epithelial sheet that closely resembled epidermis in vivo, characterized by lack of K8 expression, PCNA expression restricted to the basal layer, and K1 and filaggrin expression in the suprabasal layers. Although morphologically abnormal in many respects, some HPV-immortalized cell lines produced cornified epithelial layers and approximated the normals in their patterns of expression of keratins and filaggrin. Other HPV-immortalized cell lines produced poorly differentiated epithelial layers that were characterized by loss of filaggrin expression, and the single tumorigenic cell line, 18-11, was distinguished by abundant K8 expression. All of the HPV-immortalized cell lines were distinguished from normal keratinocytes by a common pattern of full-thickness PCNA expression in the epithelial layers they produced, suggesting that maintenance of the proliferative state may be an important contribution made by HPV 16 or 18 sequences toward the production of a malignant phenotype.     

Anti-CD3/anti-epidermal growth factor receptor-bispecific antibody retargeting of lymphocytes against human neoplastic keratinocytes in an autologous organotypic culture model.

Local cellular immune defects have been described in several tumors including human papillomavirus (HPV)-associated cervical cancer. This observation suggests the potential therapeutic benefit of immune manipulations that restore cellular immunity. Here, we evaluated the ability of bispecific monoclonal antibodies (bimAbs) to redirect T cells against keratinocytes transformed in vitro by HPV in an autologous three-dimensional culture model (organotypic cultures). The epidermal growth factor receptor (EGFR) was chosen as target for an anti-CD3/anti-EGFR bimAb because it is overexpressed in many malignant epithelial lesions and only weakly expressed in the basal layers of normal squamous epithelium. Interestingly, in organotypic cultures, the pattern of expression of EGFR was similar to that observed in vivo. The ability of T cells retargeted by CD3/EGFR bimAb to lyse HPV-transformed cell lines was confirmed in monolayer cultures. In autologous organotypic cultures, an increase in apoptotic HPV(+) keratinocytes and a significant decrease in the thickness of HPV(+) organotypic cultures were observed when activated lymphocytes and bimAbs were added to the cultures, whereas organotypic cultures of normal keratinocytes were not significantly affected. These data were similar to those obtained in the allogeneic model. These results suggest the potential usefulness of CD3-EGFR bimAb-retargeted lymphocytes in immunotherapeutic protocols for malignant epithelial lesions.     

HPV-18 confers resistance to TNF-alpha in organotypic cultures of human keratinocytes

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-alpha antiproliferative effect. The present study analyzes the effects of TNF-alpha on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-alpha. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-alpha cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-alpha. Besides, TNF-alpha treatment did not alter p16ink4a, p21cip1, p27kip1, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.     

Increased EMMPRIN (CD 147) expression during oral carcinogenesis

Gene expression profiling of oral premalignant (OPM) cells and normal oral epithelial (NOR) cells showed that EMMPRIN expression was markedly upregulated in OPM cells compared to NOR cells. We used an oral squamous cell carcinoma (OSCC) progression model composed of cell lines, organotypic cultures and tissue specimens to characterize EMMPRIN expression patterns by microarray analysis, qRT-PCR, Western blotting and immunohistochemistry. EMMPRIN levels are elevated in OPM and primary and metastatic OSCC cells as compared to NOR. EMMPRIN was detected as high and low glycosylated forms in the OPM and OSCC cellular extracts and was released in the media by OSCC cells but not by OPM cells. EMMPRIN expression in an organotypic culture model of normal and OPM mucosae mirrored the expression patterns in the respective tissues in vivo. EMMPRIN expression was limited to basal cells of normal, benign hyperkeratotic and inflammatory (lichen planus) oral mucosa. EMMPRIN expression is increased in dysplastic leukoplakias spreading to more superficial layers, and its expression levels correlated significantly with the degree of dysplasia. Primary and metastatic OSCC showed strong cell surface expression of EMMPRIN. These results suggest that EMMPRIN overexpression occurs at a very early stage of oral carcinogenesis and plays a contributing role in OSCC tumorigenesis.     

Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture

The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16(INK4A) (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the gamma2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and gamma2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and gamma2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair.     

Tannic Acid Binding of Cell Surfaces in Normal, Premalignant, and Malignant Squamous Epithelium of the Human Uterine Cervix

Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation.

While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found.

Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue.

The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules.

The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.     

Gastric Carcinomas With Unusual Cytoplasmic Ultrastructural Features

Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation.

While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found.

Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue.

The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules.

The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.     

Monoclonal antibodies specific for subsets of epidermal keratins: biochemical and immunocytochemical characterization--applications in pathology and cell culture

Keratin composition has been widely used as a biochemical marker of differentiation in normal epithelia, cell culture systems and tumours of epithelial tissues. We have been developing a model system for the study of human squamous epithelial cell differentiation, and among a panel of monoclonal antibodies we have generated for analysing this system are two antibodies recognizing subsets of epidermal keratins. The two antibodies, designated LICR-LON-16a and LICR-LON-29b, were raised to the human squamous carcinoma cell line LICR-LON-HN-5, and we describe here their biochemical and immunocytochemical characterization. Antibody 16a reacts with only epidermal basal cells in normal human skin and shows specificity for the 45 and 46 kdalton keratins. Antibody 29b stains all living layers of the epidermis, and reacts with a broad range of ketain polypeptides, (45-56 kdaltons) in immunoblotting analyses. We have investigated the alterations of cellular staining that occur in chronic hyperproliferative skin diseases and carcinomas and compared this with the staining of multilayered cultures of normal keratinocytes and the HN-5 cell line. We show that in squamous cell carcinomas and in HN-5 cell xenografts 16a and 29b stain only the well-differentiated cell types. Furthermore we found that the basal cell specificity of 16a was lost in all of the hyperproliferative skin lesions examined including psoriasis and eczema. This transition to suprabasal staining pattern was also seen in the cultures of normal keratinocytes and HN-5 cells. We conclude that aberrant keratin synthesis or abnormal post-translational processing of keratins associated with an increased rate of cell turnover could account for the altered expression of the epitope recognized by antibody 16a.     

Autocrine motility-stimulatory pathways of oral premalignant lesion cells

Patients with premalignant oral lesions have varying levels of risk of developing oral squamous cell carcinoma (OSCC), whose aggressiveness requires increased motility. Not known is if and how premalignant oral lesion cells acquire the increased motility characteristic of OSCC. This was addressed by immunohistochemical analysis of banked premalignant lesion tissues and by functional analyses using cultures established from premalignant oral lesions and OSCC. These studies showed premalignant oral lesion cells and OSCC to be more motile than normal keratinocytes. Concomitantly, levels of ceramide were reduced. The activity of the protein phosphatase PP-2A, which restricts motility and which can be activated by ceramide, was also diminished. This was due to IL-10 released from premalignant lesion cells. Treatment with a membrane-permeable ceramide restored PP-2A activity and blocked migration. These studies show an autocrine motility-stimulatory pathway that is mediated in premalignant lesion cells by IL-10 through its reduction of ceramide levels and inhibition of PP-2A activity.     

Evolution of in vitro transformation and tumorigenesis of HPV16 and HPV18 immortalized primary cervical epithelial cells.

Cervical carcinoma develops through a progressive spectrum of premalignant intraepithelial lesions (CIN I-III), the majority of which are associated with human papillomavirus (HPV) types 16 and 18. We established HPV16 and HPV18 immortalized human cervical epithelial cell lines and used them as a model to investigate the genesis and progression of cervical malignancy. The cell lines when cultured in vitro in a system mimicking their in vivo environment exhibit cytologic atypia and a variety of defects in morphologic differentiation at early passage compared to their normal counterparts. With increased passage, these alterations progress to more severe grades, histologically similar to CIN III; however only a limited number of the cell lines are tumorigenic, mimicking the epidemiologic evidence on the rate of conversion from premalignant to invasive carcinoma. The observed changes are not associated with alterations of viral DNA integration or expression and may reflect specific cellular events or changes in virus-host interactions associated with malignant progression.     

Human papillomavirus-16 and -18 replication in esophagus squamous cancer cell lines does not require sheterologous E1 and E2 proteins

Human papillomaviruses (HPVs) are doublestranded DNA viruses that replicate in the nuclei of squamous epithelial cells. HPVs can be classified into high-risk (e.g., types 16, 18, 31, and 33) or low-risk (e.g., types 6, 11, and 30), depending on their association with benign or malignant tumors. We recently described the association of HPV-16 and -18 with esophagus squamous cell cancer. HPV replication was studied in representative cell lines derived from esophagus cancers. HPV-16 and -18 genomes were independently transiently transfected into HCE-4 and HCE-7 cell lines with and without E1 and E2 genes under heterologous promoters. Southern blot analysis demonstrated that these cell lines support viral replication. However, heterologous E1 and E2 are not required for HPV replication. These findings suggest that specific host nuclear factors in esophageal squamous epithelial cells may support HPV replication. © 1995 Wiley-Liss, Inc.     

46,XY/45,X mosaicism in an amniotic fluid cell culture: suppression of abnormal cell line after subcultivation.

An abnormal cell population, 45,X, appeared in 3 of 4 cell lines established from an amniotic fluid specimen obtained from a normal mid-trimester pregnancy. Two of the cell lines were subjected to repeated chromosome analyses until VII passage. The abnormal cells were suppressed after repeated trypsinisations; simultaneously, fibroblast-like cells outgrew the cultures, which were previously predominated by epithelial-like cells. Polyploidy was found in 0 to 12% of the cells, the highest level existing in the early passages. The question of whether chromosomally abnormal cells present in primary cultures and the early subcultures reflect the karyotype of the fetus is discussed.     

Overexpression and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells.     

High frequency of human papillomavirus 6/11, 16, and 18 infections in precancerous lesions and squamous cell carcinoma of the conjunctiva in subtropical Tanzania

Dysplastic lesions and epithelial neoplasms of the conjunctiva account for approximately 2% of all malignant tumors in subtropical Tanzania. We examined the pathophysiologic role of human papillomavirus (HPV) in the development of conjunctival carcinoma in subtropical Tanzania, which has a high HPV prevalence. Tissue samples from 14 patients were obtained from the cancer registry archives at the medical center of the university in Dar es Salaam, Tanzania. A highly sensitive nonradioactive in situ hybridization technique (ImmunoMax) was applied to paraffin-embedded tissue samples to identify HPV DNA in conjunctival epithelial dysplasia and epithelial neoplasms. In each case, conventional morphologic evaluation revealed a transitional lesion extending from koilocytic dysplasia to severe dysplasia or invasive squamous cell carcinoma. Highly specific, morphologically easily distinguishable labeling of HPV-6/11, HPV-16, and HPV-18 was found in most cases. Coinfections were observed frequently. The signals showed varying intensities and different patterns of distribution. In general, higher signal intensity was found in dysplasia grades 1 and 2 and in well-differentiated areas of the invasive component of conjunctival carcinoma compared with less differentiated areas. This observation underlines the central role of HPV-16 and HPV-18 in the oncogenesis of conjunctival cancers in subtropical Tanzania.     

Three-dimensional in vitro cocultivation of lung carcinoma cells with human bronchial organ culture as a model for bronchial carcinoma.

We describe the development of a three-dimensional in vitro organ culture model for bronchial carcinoma using bronchial mucosa organ cultures and three different human non-small cell lung cancer cell lines. During precultivation, bronchial fragments obtained as biopsies during routine bronchoscopy had regenerated a complete epithelial covering with a well-preserved organotypic architecture around a nucleus consisting of connective tissue. To create cocultures, different types of confrontation between tumor cells and organ cultures were applied. Histologic light microscopy and scanning electron microscopy were used in analysis. When tumor cells were confronted with completely epithelialized organ cultures, they showed a low incidence of attachment. When organ cultures were wounded before confrontation, tumor cells always attached to the wounded side and showed a progressive invasion into the stromal tissue. Measurements of the penetration depth of tumor cells into the organ cultures after different incubation times permitted the quantitative evaluation of invasion. Histologic studies revealed well-differentiated normal epithelium in spite of long culture periods. Histologic features of the tumors were those of an invasive undifferentiated carcinoma and showed marked similarities to the situation in vivo. The coculture model permits internal controls because it contains both normal human epithelium and human tumor cells in the same organotypic culture. Therefore it offers opportunities for various in vitro investigations on therapeutic and diagnostic modalities of lung cancer, as indicated in this paper by an example of photodynamic procedures with 5-aminolevulinic acid.     

Characterization of Schwann cells from normal nerves and from neurofibromas in the bicolour damselfish

Summary Schwann cells are an important component of neurofibromas, one of the primary lesions encountered in neurofibromatosis type 1 in man. A central question in studies of neurofibromatosis type 1 has been whether the Schwann cells present in these tumours are intrinsically abnormal or exhibit abnormal phenotypes in response to stimuli from other cell types in these tumours. Damselfish neurofibromatosis is a naturally occurring disease in a species of marine fish, the bicolour damselfish, that is being developed as an animal model of neurofibromatosis type 1. Affected fish exhibit multiple neurofibromas and neurofibrosarcomas (malignant schwannomas). The present study compares the morphology, antigen expression and proliferative capacityin vitro of Schwann cells derived from peripheral nerves of normal, healthy fish with cells isolated from both spontaneously occurring and experimentally induced neurofibromas. Schwann cells from normal nerves expressed S100 antigens but not fibronectin or glial fibrillary acidic protein antigens and were similar in morphology and proliferative capacity to Schwann cells isolated from mammalian peripheral nerves. Tumour-derived cultures contained variable proportions (27–79%) of S100-positive cells that were identified as Schwann cells based on this feature. These tumour-derived Schwann cells exhibited a different morphology than normal Schwann cells, usually exhibited an increased reactivity to anti-S100 antibodies and were able to proliferate in vitro without added mitogens. Repeated subculturing of tumour-derived cultures led to the production of six cell lines all of which were composed exclusively of Schwann cells as indicated by S100 expression. These findings show that Schwann cells are an important component of tumours in Damselfish neurofibromatosis and that these cells are morphologically and physiologically altered in this disease. Observations of cell lines also suggest that tumour-derived Schwann cells are intrinsically abnormal and that this phenotype is not a result of stimuli from other cell types in the tumours.