Topographic expression of p21WAF1/SDI1/CIP1, bcl2, and p53 is altered at the early stage of colorectal carcinogenesis

We analyzed the expression of p21, bcl2, and p53 in normal and different pathologic mucosa of the human colorectum using immunohistochemistry and cold polymerase chain reaction-single strand conformation polymorphism. The topography of normal mucosa showed; bcl2 and p53 expression restricted to basal epithelial cells and p21 expressed only in superficial epithelial cells. This topographic expression was altered in hyperplastic polyps and adenomas. Hyperplastic polyps revealed absence of or weak bcl2 expression and strong p21 expression without topography. In adenomas, whereas bcl2 expression increased and extended to parabasal and superficial dysplastic epithelium, the increase of p21 expression was limited to surface dysplastic epithelium. p53 was weakly expressed throughout the full thickness of dysplastic epithelium. Bcl2 expression in adenomas was stronger than in carcinomas; p53 expression was converse and p21 expression was variable. In carcinomas, this topographic expression was largely abrogated but p53 mutation (36%) was more frequent than in adenomas (2%). In carcinomas, p21 and p53 expression correlated inversely, but there was no relationship with bcl2. These results suggest that there is precisely ordered topographic pattern of p21, bcl2, and wild p53 expression in normal colorectal cells, but this becomes disordered during the early stage of colorectal carcinogenesis.     

Modeling and validating three dimensional human normal cervix and cervical cancer tissues in vitro

The use of three dimensional in vitro systems in cancer research is a promising path for developing effective anticancer therapies. The aim of this study was to engineer a functional 3-D in vitro model of normal and cancerous cervical tissue. Normal epithelial and immortalized cervical epithelial carcinoma cell lines were used to construct 3-D artificial normal cervical and cervical cancerous tissues. De-epidermised dermis (DED) was used as a scaffold for both models. Morphological analyses were conducted by using hematoxylin and eosin staining and characteristics of the models were studied by analyzing the expression of different structural cytokeratins and differential protein marker MAX dimerisation protein 1 (Mad1) using immunohistochemical technique. Haematoxylin and eosin staining results showed that normal cervical tissue had multi epithelial layers while cancerous cervical tissue showed dysplastic changes. Immunohistochemistry staining revealed that for normal cervix model cytokeratin 10 was expressed in the upper stratified layer of the epithelium while cytokeratin 5 was expressed mainly in the middle and basal layer. Cytokeratin 19 was weakly expressed in a few basal cells. Cervical cancer model showed cytokeratin 19 expression in different epithelial layers and weak or no expression for cytokeratin 5 and cytokeratin 10. Mad1 expression was detected in some suprabasal cells. The 3-D in vitro models showed stratified epithelial layers and expressed the same types and patterns of differentiation marker proteins as seen in corresponding in vivo tissue in either normal cervical or cervical cancerous tissue. These findings imply that they can serve as functional normal and cervical cancer models.     

Integrin expression in squamous neoplasia of the cervix

Epithelial cell-basement membrane interactions are important in maintaining tissue architecture and function, and the anatomical and functional relationships between epithelial cells and their basement membranes are clearly altered in malignancy. These interactions are thought to be largely mediated by the integrins, a family of heterodimeric transmembrane glycoproteins, each consisting of an α and a β chain. Epithelial integrins mainly belong to the β1 (VLA) subfamily, which forms receptors for matrix macromolecules such as fibronectin, laminin, and collagen. There is evidence that integrin expression changes in some epithelial malignancies, possibly in relation to invasive potential. Integrin expression in cervical neoplasia was studied by immunohistochemical examination of prospectively collected colposcopic biopsies. Well-characterized monoclonal antibodies against β1–4, αl–6, and αV integrins were used to examine normal, koilocytic, and dysplastic cervical squamous epithelium, and invasive squamous carcinoma. β1, β4, α2, α3, α6, and αV were expressed by the basal layer of normal cervical squamous epithelium and by dysplastic cells in CIN (cervical intraepithelial neoplasia) 1 and 2, with none being lost and no new chains acquired. In CIN3, these integrins were either expressed throughout the ectocervical epithelium or restricted to the basal layer. In the latter cases, integrin expression was retained to a greater degree by dysplastic squamous epithelium within endocervical glands. These patterns could not be correlated with age or smear history in the cases examined. Patterns of integrin expression in neoplastic cervical epithelium therefore differ from those of normal cervical epithelium. It is possible that these changes may be related to changes in cellular function occurring during neoplastic progression.     

Argyrophilic nucleoproteins of the cervical epithelium in HPV infection and intraepithelial neoplasia]

50 colposcopic biopsies of cervical epithelium were studied, using a silver colloid technique. These comprised 28 cases of human papillomavirus infection of the cervix, 8 cases of cervical intraepithelial neoplasia (CIN) I, 8 cases of CIN II, 6 cases of CIN III. The AgNOR mean number of the basal and parabasal cells of the cervical epithelium was significantly different in virus infected cells and in CIN. Different patterns of AgNOR distribution were observed: they were single and compact in virus infection without dysplasia whereas they appeared small and often loosely arranged in dysplastic lesions. Our data suggest that this simple technique is diagnostically useful in the evaluation of borderline lesions.     

Combined Monte Carlo and finite-difference time-domain modeling for biophotonic analysis: implications on reflectance-based diagnosis of epithelial precancer

Monte Carlo (MC) modeling of photon transport in tissues is generally performed using simplified functions that only approximate the angular scattering properties of tissue constituents. However, such approximations may not be sufficient for fully characterizing tissue scatterers such as cells. Finite-difference time-domain (FDTD) modeling provides a flexible approach to compute realistic tissue phase functions that describe probability of scattering at different angles. We describe a computational framework that combines MC and FDTD modeling, and allows random sampling of scattering directions from FDTD phase functions. We carry out simulations to assess the influence of incorporating realistic FDTD phase functions on modeling spectroscopic reflectance signals obtained from normal and precancerous epithelial tissues. Simulations employ various fiber optic probe designs to analyze the sensitivity of different probe geometries to FDTD-generated phase functions. Combined MC/FDTD modeling results indicate that the form of the phase function used is an important factor in determining the reflectance profile of tissues, and detected reflectance intensity can change up to approximately 30% when a realistic FDTD phase function is used instead of an approximating function. The results presented need to be taken into account when developing photon propagation models or implementing inverse algorithms to extract optical properties from measurements.     

Scanning electron microscopy in oral cytology

Sixty-three cell smears from oral mucosa were studied by scanning electron microscopy. Among them, smears from ten healthy controls showed three kinds of cells: flat (superficial) cells with linear anastomosing microridges and microvilli; polygonal (intermediate) cells with well-defined crests between their faces and numerous microvilli; and round (parabasal) cells entirely covered by microvilli. Twenty-five smears from patients with untreated squamous-cell carcinoma showed enlarged polymorphous cells (round, globular, and elongated); microvilli, variable in their dimensions, were irregularly distributed on their surfaces. Eighteen smears from patients with severe epithelial dysplasia showed polymorphous cells with discontinuous but obvious edges separating their faces and with irregular microvilli and ridges. Nine smears were also performed in patients with various other mucosal lesions (lichen planus, leukoplakia, white sponge naevus, pemphigus vulgaris, and herpes). All of these smears were studied comparatively between examination of smears and biopsies by light microscopy. The smears were truly reliable, particularly for distinguishing between dysplastic and tumoral cells.     

Validity of the AgNOR count in cervical pathology]

Nucleolar organizer regions (NORs) were counted on ten cases each of normal ectocervix, CIN 1, 2 and 3 to verify the possibility of a differentiation between the various grades of CIN and between them and condylomata. Counts were performed on the full thickness of the tissue, layer by layer (stratified counts). A significant difference (p < 0.05) was found between the means of normal tissue toward condylomata and CIN 2 and 3 and between CIN 1 and CIN 2 and 3. There was no significance (p < 0.05) between normal tissue and CIN 1, between CIN 2 and 3 and between condylomata and CIN 2 and 3. The range of variations on the counts was associated with overlapping between the various cases. Our data showed also a progressive rise in mean NOR values from normal tissue to CIN 3. The stratified counts showed in all the groups a rise from basal to parabasal cells. Counts on parabasal and intermediate layers distinguished two groups of cases. In one there was either the same number of dots or a further rise from one layer to the next, while in the other a definite decrease was seen. The former pattern may be related to a potential for malignant evolution of the lesion. NORs should be counted in all cases of CIN and condylomata to treat more aggressively those lesions which present the pattern of a progressive rise of NORs from basal to intermediate cells.     

Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study.

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.     

Proliferation in the normal cervix and in preinvasive cervical lesions.

AIMS: To characterise further the proliferative compartment of the normal cervix and to document its alteration, if any, in the various grades of cervical intraepithelial neoplasia (CIN), particularly changes to the basal epithelial layer; to hypothesise as to the diagnostic and biological significance of any observed differences. METHOD: Proliferative compartments from 86 cervical biopsy specimens (10 normal, 11 with koilocytic change only, 12 CIN I, nine CIN II, and 44 CIN III) were determined using microwave antigen retrieval and a standard three-step Streptavidin biotin peroxidase immunocytochemical technique incorporating the MIB-1 monoclonal antibody (directed against the Ki-67 antigen). Immunoreactivity was assessed as occupying either the lower one third, lower two thirds or all three thirds of the squamous epithelium. Basal cell positivity was also quantitated. RESULTS: Specimens without CIN showed a thin suprabasal proliferative compartment two to four cells thick. True basal positivity was infrequent. With increasing grade of CIN, the growth compartment stretched evermore superficially so that in lesions of CIN III almost the full thickness of epithelium was cycling. In all grades of CIN, basal cell proliferation was significantly increased. CONCLUSIONS: In normal cervix, the parabasal layers represent the main proliferative pool with the basal layer providing a reserve. When CIN supervenes, this proliferative compartment expands commensurate with the grade of dysplasia and as basal turnover is increased specifically the intimate relation between epithelium and basement membrane might be disturbed, facilitating invasion. The diagnostic utility of these changes in growth compartments is limited.     

Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction.

The authors describe the detection of human papillomavirus (HPV) 16 DNA in paraffin-embedded, formalin-fixed tissues of cervical squamous intraepithelial lesions (SILs) by in situ hybridization after amplification by the polymerase chain reaction (PCR). Using conventional in situ hybridization and a biotin-labeled probe, variable numbers of superficial cells and none of the basal cells in the SILs showed detectable HPV 16 DNA. When the in situ assay was done after amplification, increased numbers of superficial cells had detectable HPV DNA, and the hybridization signal was much more intense. HPV DNA was also detected in basal and parabasal cells at the site of the lesion whereas not detectable in directly adjacent, normal squamous epithelium. Amplified HPV DNA was demonstrated in formalin-fixed SiHa cells using a biotin-labeled probe, demonstrating the ability to detect one copy of HPV 16 DNA. This technique should allow for direct visualization in cells of other DNA sequences of low copy number from achival specimens otherwise undetectable by conventional in situ hybridization analysis.     

Cytokeratin expression during neoplastic progression of human transitional cell carcinomas as detected by a monoclonal and a polyclonal antibody

Antibodies to intermediate filament proteins were used to study different cell layers in normal human transitional epithelium, 16 human transitional cell carcinomas, and two cell lines derived from human bladder carcinomas. Conventional rabbit antisera to human skin keratins stained all layers of the transitional epithelium from bladder, ureter, and kidney. A slightly higher staining intensity was found in the basal and superficial layers as compared with the intermediate cell layers. A monoclonal antibody to cytokeratin 18 (RGE 53), however, stained only the superficial cell layer of transitional epithelium, the so-called umbrella cells. In well-differentiated (grade I) transitional cell carcinomas, RGE 53 stained only the superficial cells of papillary structures. In higher grade papillary tumors, RGE 53 also stained cells within the basal and intermediate layers, whereas in high-grade, invasive tumors almost all tumor cells were RGE 53 positive. These results show that monoclonal antibodies to cytokeratins can provide both an indication of processes involved in neoplastic progression of bladder tumors and a means of studying the molecular relationship of the tumor cells to normal cells.     

Premalignant and malignant oral lesions are associated with changes in the glycosylation pattern of carbohydrates related to ABH blood group antigens

The distribution of carbohydrate structures related to the ABO(H) blood group antigen system was studied in biopsies from eight squamous cell carcinomas, and eight erythroplakias with epithelial dysplasia. Twenty oral lesions without histological evidence of malignancy (13 lichen planus lesions and 7 homogeneous leukoplakias) were also examined. The distribution of Lex, Ley, H type 2 chain, and N-acetyllactosamine, all type 2 chain carbohydrate structures, was investigated by immunohistological staining using monoclonal antibodies with selected specificity. The histological pattern of expression of these antigens in the benign lesions was similar to that of normal oral mucosa, i.e. expression of: N-acetyllactosamine on basal cells, H antigen on parabasal cells, and Lex and Ley on spinous cells. However, lesions with epithelial dysplasia showed H antigen on all spinous cells, and often also on basal cells, with expression of Lex and Ley restricted to the most superficial part of the epithelium above the H-positive cell layers. In carcinomas most cells were negative for H antigen but were positive for Ley and Lex in 5 out of 8 cases.     

Immunohistochemical localization of syndecan-1 in normal and pathological human uterine cervix

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.     

Demonstration of an epitope of the transferrin receptor in human cervical epithelium--a potentially useful cell marker.

The distribution of an epitope of the transferrin receptor in the human uterine cervical epithelium has been investigated. Immunohistochemical staining, both immunofluorescent and immunoperoxidase, was performed on biopsy specimens and cytological samples from normal, dysplastic, and neoplastic cervical epithelia using the monoclonal OKT9 antibody. The results of staining 145 cervical biopsy specimens with OKT9 showed widespread staining in all malignant epithelia and most severely dysplastic epithelia. No such staining was seen in either normal epithelia or in mildly dysplastic epithelia apart from the staining of the basal cell layer in some normal epithelia. The incidence of staining in the 50 cervical cytocentrifuge preparations was not as high as that in the 145 tissue sections. The potential role of the OKT9 antibody in both the screening of cervical cytocentrifuge preparations and the prediction of malignancy is discussed. The antibody is considered to be of more value in the examination of biopsy material than of cytocentrifuge preparations.     

Expression of the complement regulatory proteins decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 in the normal human uterine cervix and in premalignant and malignant cervical disease.

The membrane-bound complement regulators decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), and CD59 are broadly expressed proteins that act together to protect host tissues from autologous complement. Comparison of expression profiles of these proteins between normal and pathological tissues could reveal a mechanism by which tumor cells evade complement-mediated killing. Expression of the regulators was therefore examined in the normal human uterine cervix, in cervical intraepithelial neoplasia (CIN; n = 23), and in cervical squamous carcinomas (n = 6). DAF and MCP were reciprocally expressed in normal ectocervical epithelium. MCP was confined predominantly to the basal and parabasal layers with more extensive expression in metaplastic squamous epithelium. An apparent expansion in MCP expression was observed in more severe premalignant lesions whereas cervical carcinoma were uniformly MCP positive. By contrast, DAF expression appeared unaltered in premalignant lesions and variable in carcinomas. However, increased DAF was observed in stromal cells directly adjacent to infiltrating tumor cells. A low molecular weight DAF product was detected in tumors, and preliminary evidence suggests this may be derived from stromal cells. Overall, changes in expression of C3 convertase regulators in both the stromal and epithelial compartments may be important for evasion of immune surveillance in cervical cancer.     

Adhesion of Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis to bovine urinary bladder epithelial cells of various ages and levels of differentiation.

The adhesion of Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis to various epithelial cell layers of bovine urinary bladders was examined. Adhesion was most efficient to the urinary sediment epithelial cells and the superficial cells immediately before shedding, followed by the remaining superficial cells and intermediate cells in this order, and least efficient to the deeper intermediate and basal cells. Incubation of the intermediate cells for 6 h increased the number of bacteria that adhered to these cells.     

Far-field superposition method for three-dimensional computation of light scattering from multiple cells

A linear coherent superposition method for estimating the plane wave far-field scattering pattern from multiple biological cells computed by the finite-difference time-domain (FDTD) method is presented. The method enables the FDTD simulation results of scattering from a small number of complex scatterers, such as biological cells, to be used to estimate the far-field pattern from a large group of those same scatterers. The superposition method can be used to reduce the computational cost of FDTD simulations by enabling a single large scattering problem to be broken into smaller problems with more practical computational requirements. It is found that the method works best in cases where there is little multiple scattering interaction between adjacent cells, so the far-field pattern of multicell geometry can simply be calculated as a phase-adjusted linear superposition of the scattering from individual cells. A strategy is also presented for choosing the minimum number of cells in cases with significant multiple scattering interactions between cells.