Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: an immunohistochemistry study

Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331microM BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10(8) nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10(8) nucleotides, with a median of 75/10(8) nucleotides. PAH-DNA adduct values above 150/10(8) nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear related to the increased risk of cervical precancer and cancer among carcinogenic HPV-infected smokers.     

Expression of retinoblastoma gene product in respiratory epithelium and sinonasal neoplasms: relationship with p16 and cyclin D1 expression

Transition from G1 to S phase of the cell cycle is mediated by interactions between the Retinoblastoma gene product (pRb), p16, and cyclin D1. To determine the expression of these proteins in the sinonasal mucosa immunohistochemistry was carried out on archived tissue sections from 46 patients (37 men, 9 women, age range 17 to 82 years, median 55 years). Nuclear immunostaining for these proteins was assessed and the expression rates (percentages of immunoreactive nuclei) in normal respiratory epithelium, inverted sinonasal papillomas, cylindrical (oncocytic) sinonasal papillomas, and squamous cell carcinomas were compared. Normal respiratory epithelium showed significantly higher pRb expression in surface cells compared to basal cells (p < 0.05). In contrast, abundant pRb expression in surface and basal cells was detected in columnar differentiation in sinonasal papillomas and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed significantly reduced pRb expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05, respectively). Expression of p16 was detected in all epithelial cell layers of normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed increased p16 expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05 between squamous metaplasia and columnar epithelium). Sinonasal squamous cell carcinomas showed the coexpression of pRb and p16. Expression rates of cyclin D1 higher than 10% were detected only in invasive carcinomas but not in carcinoma in situ, sinonasal papillomas or respiratory epithelium. Conclusively, pRb expression accompanies terminal differentiation in columnar surface cells. Expression of pRb in proliferating basal cells is present in sinonasal papillomas and adjacent mucosa but not in normal respiratory epithelium. Cuboidal and squamous metaplasia in inverted papillomas involves downregulation of pRb expression along with increased p16 expression in surface cells. Sinonasal squamous cell carcinomas coexpress pRb and p16. Overexpression of cyclin D1 in sinonasal lesions is confined to invasive squamous cell carcinomas.     

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells from a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.     

Regeneration of vitamin A deficient rat tracheal epithelium after mild mechanical injury. Growth kinetics and cellular differentiation.

Mild mechanical abrasion of tracheal epithelium of Vitamin A deficient rats removed the superficial cells and spared basal cells which divided to repopulate the damaged area. The proliferative cells passed through a period of DNA synthesis with the greatest numbers of thymidine incorporating cells in samples labelled 22 h after injury. A peak of cell division occurred at 32 h and there was no further DNA synthesis or cell division. The area of wounding exhibited squamous metaplasia while normal pseudostratified muco-ciliary structure was retained by adjacent epithelium which had not been injured. The data indicates that squamous metaplasia in the respiratory epithelium in longstanding Vitamin A deficiency is due to redirected differentiation of basal cells and is seen only after mitotic activity has occurred.     

Regeneration of Vitamin A Deficient Rat Tracheal Epithelium After Mild Mechanical Injury

Mild mechanical abrasion of tracheal epithelium of Vitamin A deficient rats removed the superficial cells and spared basal cells which divided to repopulate the damaged area. The proliferative cells passed through a period of DNA synthesis with the greatest numbers of thymidine incorporating cells in samples labelled 22 h after injury. A peak of cell division occurred at 32 h and there was no further DNA synthesis or cell division. The area of wounding exhibited squamous metaplasia while normal pseudostratified muco-ciliary structure was retained by adjacent epithelium which had not been injured. The data indicates that squamous metaplasia in the respiratory epithelium in longstanding Vitamin A deficiency is due to redirected differentiation of basal cells and is seen only after mitotic activity has occurred.     

PAH-DNA adducts in a Chinese population: relationship to PAH exposure, smoking and polymorphisms of metabolic and DNA repair genes.

The present study was conducted in a Chinese population to evaluate the usefulness and sensitivity of PAH-DNA adduct as a biomarker of PAH exposure, and to examine the potential effects of smoking and polymorphisms of responsive genes on DNA adduct formation induced by PAH exposure. The polymorphisms of genes examined include GSTM1, GSTT1, CYP1A1, microsomal epoxide hydrolase (mEH) and excision repair cross-complementary group 2 (ERCC2). A total of 194 subjects with a broad range of PAH exposures were recruited, including 116 occupationally exposed workers, 49 metropolitan residents and 29 suburban gardeners. A significant exposure-response relationship was observed between PAH exposure and DNA adducts in leukocytes across the entire group of subjects (p < 0.0001). The levels of PAH-DNA adducts in the subgroup with lowest occupational exposure to PAHs (< 0.1 microg BaP m(-3)) was significantly higher than that in metropolitan residents and suburban gardeners. However, no significant difference was detected between residents and gardeners, with mean BaP concentrations of 0.028 and 0.011 microg m(-3), respectively. The polymorphisms of genes examined failed to show significant effects on PAH-induced adduct formation except ERCC2 Lys751Gln genotypes. A significantly higher level of PAH-DNA adduct was found in subjects with wild-type ERCC2 than those who have either heterozygous or homozygous variant alleles (p < 0.01). Smoking, age and gender did not substantially contribute to PAH-induced DNA adduct formation in this study. The study suggests that PAH-DNA adducts may serve as a reliable biomarker of PAH exposure in occupational settings but may not be sensitive enough to be used in populations with environmental exposures to PAHs.     

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 10⁸ unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the ³²P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.     

Enrichment of cysteinyl adducts of human serum albumin

We report a method to enrich cysteinyl adducts of human serum albumin (HSA), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys34, we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples, with abundances decreasing in the following order: commercial HSA > archived HSA > fresh HSA. After enrichment of HSA, mercaptalbumin was no longer observed in mass spectra. The ratios of HSA adducts post-/preenrichment, quantified via the Bradford assay and gel electrophoresis, were 0.029 mg adducts/mg HSA in fresh HSA and 0.323 mg adducts/mg HSA in archived HSA. The apparent elevation of adduct levels in archived samples could be due to differences in specimen preparation and storage rather than to differences in circulating HSA adducts. We conclude that thiol-affinity resins can efficiently remove mercaptalbumin from HSA samples prior to characterization and quantitation of protein adducts of reactive systemic electrophiles.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs . In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC , as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs. In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC, as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Discrimination of various epithelia by simple morphometric evaluation of the basal cell layer. A light microscopic analysis of pseudostratified, metaplastic and dysplastic nasal epithelium in nickel workers

This study presents a simple morphometric method for objective classification of pseudostratified, various types of metaplastic, and dysplastic epithelium by evaluation of cellular features in the basal layer only. Fifty-four biopsy specimens were taken for diagnostic reasons from the nasal mucosa of nickel workers, and semithin toluidine-blue-stained sections were analysed. The most sensitive parameters in distinguishing between the various types of epithelium were: (i) the transverse nuclear diameter, (ii) the size of the nucleoli and (iii) the basal cell width expressed as an index weighted towards the cell profiles with the broadest attachment face on the basal lamina. A combination of these three parameters allows a clear separation between dysplastic, metaplastic and pseudostratified epithelium. The sequential increase in these parameters from pseudostratified epithelium through two histologically distinguishable stages of metaplasia (stratified cuboidal and mixed stratified cuboidal/stratified squamous epithelium) to fully developed squamous epithelium supports the concept that metaplasia develops gradually. The continuous increase in these parameters from metaplasia to dysplasia further suggests that metaplasia is a necessary step in the development of nasal epithelial dysplasia. This morphometric model appears especially useful in monitoring small sequential epithelial changes, and might also be used for evaluating other types of epithelia.     

声带组织切片方法的比较

目的:探讨犬声带冠状位切片与水平位切片各自的特点,为声带实验提供合适的切片方法。方法:家犬4只,2只取材后行冠状位石蜡切片,2只取材后行水平位石蜡切片。通过HE染色观察声带固有层的一般组织结构,Masson三色染色观察固有层中胶原的排列情况。结果:HE染色示冠状位、水平位切片均可见声带表面被覆复层鳞状上皮,固有层内有大量排列紧密的纤维组织,纤维组织中夹杂少量腺体,固有层下方为肌层。冠状位切片可观察声带某一点冠状面固有层的情况,若观察整个声带的情况需声带连续切片;水平位切片可在一张切片中观察到前联合、声带膜部及声带突部位的固有层情况,解剖标志明显,利于定位。Masson三色染色示冠状位、水平位切片均可见固有层浅层有较细的胶原纤维束,中层有较粗的纤维束与较细的纤维束交织排列,深层纤维束排列更紧密。结论:冠状位切片可观察声带某一点冠状面固有层的整体情况,水平位切片可在一张切片中观察到前联合、膜部及声带突部位的固有层情况。     

Osteopontin's colocalization with the adhesion molecule CEACAM5 in cytoplasm of carcinoma of tongue and its correlation with the invasion of that diease

ABSTRACT: The purpose of this study was to investigate the expression of carcinoembryonic antigenrelated cell adhesion molecule 5 (CEACAM5) and correlate it with OPN expression and function in squamous carcinoma of tongue. Paraffin were sections of 80 samples with squamous carcinoma of tongue and 40 samples with normal tissue of tongue for benign lesion having undergone surgery. Immunohistochemistry (IHC) was used to study the distribution of CEACAM5 and OPN, and double-labeling immunohistochemistry was used to observe the relationship between CEACAM5 and OPN expression. CEACAM5 and OPN are found in normal tissue of tongue, but with different expression pattern. CEACAM5 expression mainly with membranous staining is restricted on the superficial epithelium. However, OPN expression with mainly cytoplasmic staining is restricted on the deep epithelium. No colocalization of CEACAM5 and OPN have been observed in normal tissue of tongue. In squamous carcinoma of tongue, CEACAM5 expression with cytoplasmic staining is different from normal tongue tissue with membranous staining, and the transformation of CEACAM5 distribution from membrane to cytoplasm is an important incident for the invasion and differentiation of tumor. CEACAM5 and OPN are colocalized in cytoplasm, and a significant correlation was observed between the positive colocalization and the negative colocalization in the depth of invasion and the differentiation of the tumor.     

PAH-DNA adducts in a Chinese population: relationship to PAH exposure,smoking and polymorphisms of metabolic and DNA repair genes

Abstract

The present study was conducted in a Chinese population to evaluate the usefulness and sensitivity of PAH-DNA adduct as a biomarker of PAH exposure, and to examine the potential effects of smoking and polymorphisms of responsive genes on DNA adduct formation induced by PAH exposure. The polymorphisms of genes examined include GSTM1, GSTT1, CYP1A1, microsomal epoxide hydrolase (mEH) and excision repair cross-complementary group 2 (ERCC2). A total of 194 subjects with a broad range of PAH exposures were recruited, including 116 occupationally exposed workers, 49 metropolitan residents and 29 suburban gardeners. A significant exposure–response relationship was observed between PAH exposure and DNA adducts in leukocytes across the entire group of subjects (p<0.0001). The levels of PAH-DNA adducts in the subgroup with lowest occupational exposure to PAHs (<0.1µg BaP m^?3) was significantly higher than that in metropolitan residents and suburban gardeners. However, no significant difference was detected between residents and gardeners, with mean BaP concentrations of 0.028 and 0.011µg m^?3, respectively. The polymorphisms of genes examined failed to show significant effects on PAH-induced adduct formation except ERCC2 Lys751Gln genotypes. A significantly higher level of PAH-DNA adduct was found in subjects with wild-type ERCC2 than those who have either heterozygous or homozygous variant alleles (p<0.01). Smoking, age and gender did not substantially contribute to PAH-induced DNA adduct formation in this study. The study suggests that PAH-DNA adducts may serve as a reliable biomarker of PAH exposure in occupational settings but may not be sensitive enough to be used in populations with environmental exposures to PAHs.     

Immunoperoxidase detection of 4-aminobiphenyl- and polycyclic aromatic hydrocarbons-DNA adducts in induced sputum of smokers and non-smokers

Tobacco smoke constituents, 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAH) possess carcinogenic properties as their reactive metabolites form DNA adducts. We studied the formation of 4-ABP- and PAH-DNA adducts in induced sputum, a non-invasively obtainable matrix from the lower respiratory tract, of smokers (n=20) and non-smokers (n=24) utilizing a semi-quantitative immunohistochemical peroxidase assay. Smokers had significantly higher levels of 4-ABP-DNA adducts as compared to non-smokers (0. 08+/-0.02 versus 0.04+/-0.01, P=0.001, density of immunohistochemical staining), and the levels of adducts were related to current smoking indices (cigarettes/day: r=0.3, P=0.04 and tar/day: r=0.4, P=0.02). Likewise, smokers had elevated levels of PAH-DNA adducts as compared to non-smokers, however, the differences was not statistically significant (0.13+/-0.02 versus 0. 08+/-0.02, P=0.07). The levels of PAH-DNA adducts were only significantly related to the amount of tar consumed per day (r=0.4, P=0.04) but not to the number of cigarettes smoked per day. Neither the levels of 4-ABP-DNA adducts nor those of PAH-DNA adducts were related to smoking history index (pack years). Further, the levels of 4-ABP-DNA adducts were correlated with those of PAH-DNA adducts (r=0.4, P=0.02). We conclude that immunohistochemistry of 4-ABP-DNA adducts in induced sputum is a specific approach to assess current exposure to tobacco smoke in the lower respiratory tract, however, in the case of PAH-DNA adducts, such analysis is less specific as it does not explicitly reflect the magnitude of the exposure.     

Specific receptor detection by a functional keratinocyte growth factor- immunoglobulin chimera

Fibroblast growth factor receptors (FGFRs) are encoded by at least four distinct highly conserved genes, and alternative splicing generates multiple gene products. The close relationship among different FGFRs has greatly increased the difficulty in generating specific immunochemical probes. As an alternative strategy, we constructed a fusion protein comprising keratinocyte growth factor (KGF) and an IgG1 Fc domain (HFc). The chimeric molecule was efficiently secreted from transfectants as a disulfide-linked dimer that bound KGFRs with high affinity. Moreover, the KGF-HFc, like native KGF, induced DNA synthesis by epithelial cells implying normal functional receptor activation. Because it retained the convenient detection properties of an immunoglobulin, it was possible to use the KGF-HFc in ligand-mediated histochemical analysis of KGFRs. Flow cytometry revealed KGF-HFc chimera detection of the KGFR, an alternative FGFR2 product, but not FGFR1 (flg) or FGFR2 (bek). Histochemical analysis of normal skin demonstrated the specific localization of KGFRs within the spinous layer, a zone of epithelial cell differentiation. KGFRs were also localized to epithelial cells within a specific region of the hair follicle, and they were not detectable in cells of the sweat gland. Tissue sections of soft palate and tonsil, two examples of nonkeratinizing epithelium, revealed staining of stratum spinosum and some staining of the basal cell layer as well. Neither salivary gland epithelium nor lymphoid cells were positive. The ciliated epithelium of the trachea exhibited KGFR expression in intermediate and basal cell layers. In striking contrast to the normal pattern of staining in the adjacent epithelium, a squamous cell carcinoma of skin lacked detectable KGFRs. Our present findings suggest that growth factor-Ig fusion proteins may be generally applicable in ligand-mediated histochemical detection and localization of growth factor receptors.     

Methyl green-pyronin stain distinguishes proliferating from differentiated nonproliferating cell nuclei after acid denaturation of DNA

We applied methyl green-pyronin (MG-P) stain, which is usually used for the selective staining of DNA and RNA, to frozen sections of rat jejunal and esophageal mucosa, following digestion with RNase and treatment with various concentrations of HCl. The pyroninophilia of the nuclei increased with increasing strength of the acid, but the susceptibility of the nuclei to acid differed among cell populations. In the jejunal epithelium, at an appropriate acid strength the nuclei in the crypts of Lieberkuhn were less acid-sensitive and remained blue-green, whereas those in the villi were more pyroninophilic and stained lavender. Under the same conditions, the nuclei in the basal layer of the esophageal epithelium were blue-green and those in the spinous and granular layers were increasingly lavender. These results suggest that in cell-renewal systems the differentiated, nonproliferating cells are more sensitive to acid denaturation of DNA than the undifferentiated, actively proliferating cells. MG-P stain, which is able to distinguish double-stranded from single-stranded DNA, may be used as a tool to stain proliferating and nonproliferating cell nuclei differentially in tissue sections.     

Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.