Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.     

Nucleolar organizer regions in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The number of nucleolar organizer regions (NORs) stained by the one-step silver colloid method was measured in preneoplastic and neoplastic bladder lesions induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. Male ACI/N rats, 6 weeks of age, were given 0.05% BBN in drinking water for 5, 8, 12, 18 and 30 weeks to induce preneoplastic and neoplastic transitional cell lesions. The mean numbers of silver-stained NORs (AgNORs) in such lesions were as follows: untreated transitional epithelium (n = 6), 1.26 +/- 0.09; transitional cell epithelium outside focal lesions (n = 10), 1.75 +/- 0.10; simple hyperplasia (n = 10), 2.01 +/- 0.15; papillary or nodular (PN) hyperplasia (n = 10), 2.15 +/- 0.19; transitional cell papilloma (n = 5), 2.37 +/- 0.12; transitional cell carcinoma (n = 5), 3.52 +/- 0.23. Thus, the mean number of AgNORs showed a step-wise increase from untreated and treated, histologically normal transitional epithelium through simple hyperplasia and PN hyperplasia to transitional cell papilloma and carcinoma. These results suggest that the mean number of AgNORs may reflect the proliferative nature of bladder lesions induced by BBN, as reported in preneoplastic and neoplastic lesions in other organs. PN hyperplasias were classified into two types based upon the mean number of AgNORs, indicating that they include reversible and irreversible changes in contrast with simple hyperplasia which is reversible change.     

Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of γ-glutamyltranspeptidase activity

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of γ-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20–25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H₂O₂ plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent path-way of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.     

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Isolation of a high spin form of cytochrome P-450 induced in rat liver by 3-methylcholanthrene

A form of cytochrome P-450 (P-450 MC₁) has been isolated from the livers of 3-methylcholanthrene-treated rats. The molecular weight is 54,500 and the heme iron is in the high spin configuration which clearly differenciates this form from the other major cytochrome induced by 3-methylcholanthrene (P-450 MC₂). Whilst MC₂ actively dealkylated 7-ethoxycoumarin and 7-ethoxyresorufin, MC₁ was only active with 7-ethoxyresorufin. Ouchterlony immunodiffusion analysis and ELISA showed that anti MC₁ and anti MC₂ reacted with both MC₁ and MC₂ but preferentially with the homologous antigen. Both anti MC₁ and MC₂ cross-reacted strongly with microsomes from 3-methylcholanthrene, Aroclor 1254 and isosafrole-treated rats and also, but much weaker, with microsomes from phenobarbital, $\text{trans}$-stilbene oxide and chlofibrate-treated as well as untreated rats. Both MC₁ and MC₂ are induced by the same inducers, 3-methylcholanthrene, Aroclor 1254 and also isosafrole, whilst phenobarbital, $\text{trans}$-stilbene oxide and chlofibrate did not induce either of them, which shows that MC₁ and MC₂ are under similar control by various types of inducers, but MC₁ was present in control microsomes at higher levels than MC₂.     

Immunocytochemistry of collagenase expression in transitional cell carcinoma of the bladder

OBJECTIVE: To evaluate the usefulness of collagenase immunocytochemistry as well as its immunohistochemistry in assessing the correlation with prognostic factors in transitional cell carcinoma (TCC) of the urinary bladder. STUDY DESIGN: We investigated the expression of collagenase in catheterized urine and histologic specimens from 38 patients with TCC and 20 cases with benign lesions of the urinary tract. RESULTS: Thirteen (34.2%) and 17 (44.7%) patients with TCC showed positive expression of collagenase on cytologic and histologic specimens, respectively, whereas in no cases with benign lesions was such expression found (P < .01). Invasive and nonpapillary TCC had higher positive rates than noninvasive and papillary TCC. Grade 3 TCC was positive at a higher rate than was grade 2, whereas there were no positive cases with grade 1. Collagenase expression did not correlate significantly with stage. CONCLUSION: Collagenase expression in urinary TCC correlated well with tumor growth pattern, pathologic grade and invasiveness of the carcinoma; all are known to be prognostic factors. The application of collagenase immunostaining to urinary cytology is very useful for assessing prognosis in TCC.     

Aflatoxin induced hepatocarcinogenesis in ducks

To assess the carcinogenic response of duck hepatic tissue, an experimental study was undertaken. Pure aflatoxin B₁, was administered to twelve white pekin ducks of two to three months of age at a dose rate of 0.04165 mg/kg body weight every third day for six months. There was reduction in body weight and haemoglobin level from the third month onwards. Total serum protein, albumin and globulin had a slow and gradual reduction and ESR was significantly increased from the third month. Serum enzymes (AST, ALT, GGT and ALP) were significantly increased from the Vlllth to XVth fortnights (Two weeks). Ten ducks developed hepatic tumours by 180th day. Four of them had neoplastic nodules on the 90th day. Histo-pathologically they were hepatocellular carcinoma (6), Cholangiocellular carcinoma (4) and Chronic hepatitis (2). There was moderate to severe expression of GGT and ALP in the liver tissue during neoplastic transformation.     

Expression of multiple proteins structurally related to gamma-glutamyl transpeptidase in non-neoplastic adult rat hepatocytes in vivo and in culture.

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma-glutamyl transpeptidase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct-ligated rats. However, the induction of GGT activity in rat hepatocytes by two-thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross-react with 2 different anti-rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9- and 58.0-kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5-kDa protein represents 50% of the total GGT-immunoreactive protein, in contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5-kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79-kDa single-chain glycoprotein propeptide of GGT. It appears that in normal hepatocytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.     

Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae

Summary We have investigated single and combined effects of calciotropic hormones and growth factors on the regulation of alkaline phosphatase (ALP) activity and calcium metabolism in an optimized serum-free bone organ culture system of embryonic chick tibiae. Parathyroid hormone PTH(1–34) alone mobilized calcium from bone tissue time- and dose-dependently and inhibited ALP activity. Both the bisphosphonate (BM 21.0955) and to a lesser extent salmon calcitonin alone slightly increased calcium uptake and inhibited the stimulation of bone resorption by PTH(1–34). 1,25(OH)₂D₃ mobilized calcium and inhibited ALP activity in contrast to 24,25(OH)₂D₃ which inhibited ALP activity but had no significant effect on calcium metabolism. Interestingly the combination of PTH(1–34) with 1,25(OH)₂D₃ but not 24,25(OH)₂D₃ reduced calcium mobilization. The combination of the midregional fragment PTH(28–48), which by itself has no effect on calcium metabolism, with 1,25(OH)₂D₃ reduced calcium mobilization more efficiently. Several PTH-regulated mediators have been assayed in this system. Of the tested growth factors, IGF-I at high concentrations caused bone resorption with no effect on ALP activity. TGF-β₁ (transforming growth factor β) and BMP-2 had no significant effect on calcium metabolism; however, ALP activity was inhibited by TGF-β₁ and induced dose dependently by BMP-2. Of the other factors known to be present in bone, platelet-derived growth factor (PDGF_A/B) and epidermal growth factor (EGF) had a small effect on calcium mobilization but had no effect on ALP activity. bFGF reduced ALP activity slightly without an effect on calcium metabolism. Our results show that this in vitro system can mimic some interactions of calciotropic hormones in vivo and allows the assaying of mediators in terms of regulation of ALP activity and of calcium metabolism.     

Distribution of glucose-6-phosphatase activity in normal,hyperplastic, and preneoplastic rat liver

The significance of glucose-6-phosphatase (G6P) expression by bile duct-like cells proliferating during hepatocarcinogenesis in the histogenesis of hepatocellular carcinoma is not clear. To this end, we measured the histochemical and biochemical activity of G6P in normal rat liver, and in rat livers in which bile duct-like proliferation was induced by either hyperplastic (bile duct ligation for 14 days or feeding alpha-naphthylisothiocyanate for 28 days) or neoplastic (feeding a choline-devoid diet containing 0.1% ethionine for 60 days) regimens. In normal, hyperplastic, and preneoplastic livers, G6P histochemical activity was confined to the hepatocytes; proliferated bile duct-like cells, like normal bile ducts, did not display visible G6P staining. When the enzyme activity was determined biochemically, however, hydrolysis of glucose-6-phosphate was observed in both parenchymal and nonparenchymal liver cells isolated from all experimental animals. In elutriated nonparenchymal fractions, G6P activity was directly proportional to the number of cells positive for gamma-glutamyl transpeptidase and cytokeratin no. 19 (markers of bile duct cells) and inversely proportional to the number of cells positive for vimentin (marker of mesenchymal cells). These results indicate that, while by light microscopy hepatic G6P histochemical activity is detectable only in the hepatocytes, the biochemical activity is also expressed in proliferating bile duct-like cells. However, the nonparenchymal activity is observed during both neoplastic and hyperplastic liver growth, thus indicating that the presence of this enzyme in bile duct-like cells proliferating during hepatocarcinogenesis should not necessarily be construed as supporting their stem cell nature nor their neoplastic commitment.     

Effect of phenobarbital and the peroxisome proliferator ciprofibrate on γ-glutamyltranspeptidase activity and leukotriene C4 concentration in cultured rat hepatocytes

Peroxisome proliferators induce hepatocellular carcinomas in rodents by an unknown mechanism. γ-Glutamyltranspeptidase (GGT), a biochemical marker for identifying putative preneoplastic lesions in the liver, is highly expressed in phenobarbital (PB)-promoted altered hepatic foci but not in those promoted by peroxisome proliferators. One of the substrates of GGT is the eicosanoid LTC₄. Because peroxisome proliferators and PB have differing effects on eicosanoid metabolism in vivo, we hypothesized that PB would similarly increase LTC₄ concentrations, whereas the peroxisome proliferator ciprofibrate (CIP) would not. Cultured hepatocytes were treated with the peroxisome proliferator ciprofibrate (CIP: 100 and 400 μM) or PB (PB: 0.5 and 2 mM). Competitive radioimmunoassay (RIA) was used to determine the concentration of LTC₄ in extracts of cultured hepatocytes. CIP decreased the concentration of LTC₄ throughout the culture period, but PB increased the LTC₄ concentration. Both doses of CIP significantly inhibited the induction of GGT activity at 48 and 72 hours, whereas PB enhanced GGT activity. We therefore hypothesized that LTC₄, a substrate of GGT, may induce GGT activity. LTC₄, however, did not enhance GGT activity and inhibited it at very high concentrations. The results of this experiment show that CIP and PB have different effects on GGT activity and LTC₄ concentration. LTC₄, however, does not induce GGT in cultured hepatocytes. © 1996 John Wiley & Sons, Inc.     

Distribution of glucose-6-phosphatase activity in normal, hyperplastic, and preneoplastic rat liver.

The significance of glucose-6-phosphatase (G6P) expression by bile duct-like cells proliferating during hepatocarcinogenesis in the histogenesis of hepatocellular carcinoma is not clear. To this end, we measured the histochemical and biochemical activity of G6P in normal rat liver, and in rat livers in which bile duct-like proliferation was induced by either hyperplastic (bile duct ligation for 14 days or feeding alpha-naphthylisothiocyanate for 28 days) or neoplastic (feeding a choline-devoid diet containing 0.1% ethionine for 60 days) regimens. In normal, hyperplastic, and preneoplastic livers, G6P histochemical activity was confined to the hepatocytes; proliferated bile duct-like cells, like normal bile ducts, did not display visible G6P staining. When the enzyme activity was determined biochemically, however, hydrolysis of glucose-6-phosphate was observed in both parenchymal and nonparenchymal liver cells isolated from all experimental animals. In elutriated nonparenchymal fractions, G6P activity was directly proportional to the number of cells positive for gamma-glutamyl transpeptidase and cytokeratin no. 19 (markers of bile duct cells) and inversely proportional to the number of cells positive for vimentin (marker of mesenchymal cells). These results indicate that, while by light microscopy hepatic G6P histochemical activity is detectable only in the hepatocytes, the biochemical activity is also expressed in proliferating bile duct-like cells. However, the nonparenchymal activity is observed during both neoplastic and hyperplastic liver growth, thus indicating that the presence of this enzyme in bile duct-like cells proliferating during hepatocarcinogenesis should not necessarily be construed as supporting their stem cell nature nor their neoplastic commitment.     

Colorectal carcinoma and its precursors: role of argyrophilic nucleolar organizer regions (AgNORs).

A silver colloid technique to identify Argyrophilic Organizer Region (AgNOR) was applied to 5 hyperplastic polyps, 5 adenomas with low grade dysplasia, 5 adenomas with high grade dysplasia and 15 adenocarcinomas of the large bowel (5 well differentiated, 5 moderately differentiated and 5 poorly differentiated). The authors suggest the plainness and usefulness of simultaneous application of clumps per cell, AgNORs per clump and total AgNORs counts in the evaluation of neoplastic and preneoplastic lesions of the colon. In fact the results show that the number of clumps per cell is useful to distinguish hyperplastic polyps from adenomas with high grade dysplasia and from all the adenocarcinomas. Using the number of AgNORs per clump there is significant difference between hyperplastic polyps and adenomas with high grade dysplasia and between adenomas with low grade dysplasia and well differentiated adenocarcinomas. Finally the total number of AgNORs can discriminate hyperplastic polyps from adenomas with high grade dysplasia and these from adenomas with low grade dysplasia.     

Quantitative microscopy of human DNA topoisomerase II-alpha expression in transitional cell carcinoma of the bladder

OBJECTIVE: To compare the nuclear size of various grades of transitional cell carcinoma (TCC) stained immunohistochemically with the nuclear enzyme topoisomerase II-alpha (topo II-alpha) in bladder urothelial neoplasms. STUDY DESIGN: Histologic sections from 53 consecutive papillary bladder neoplasms were stained immunohistochemically for topo II-alpha expression. There were 18 (33.9%) urothelial neoplasms of low malignant potential (UNLMP), 18 (33.9%) low grade urothelial carcinoma (LGUCa), and 17 (32%) with high grade urothelial carcinoma (HGUCa). The histologic slides were photographed at 400 x magnification and then projected on a screen, and the area with stained nuclei was measured. RESULTS: The cells and nuclei in HGUCa were significantly larger than in LGUCa (P < .05) and UNLMP (P < .01). CONCLUSION: Calculation of the area fraction of nuclei in TCC of the bladder stained with topo II-alpha is an additional method of establishing the grade of these tumors.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

Different requirement for cytochrome b5 in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450

The role of cytochrome b₅ in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450_PB and P-450_MC) purified from rat liver microsomes. The O-deethylation by P-450_PB absolutely required the presence of cytochrome b₅, whereas the same reaction catalyzed by P-450_MC did not require cytochrome b₅. These effects of cytochrome b₅ on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b₅. On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b₅. These observations suggest that the requirement for cytochrome b₅ in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized.     

Distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa: age-related progression

Age-related changes were examined in the distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa. The olfactory mucosa of female ICR mice at postnatal ages from 10 days to 16 months were investigated histologically by hematoxylin and eosin staining, high-iron diamine-Alcian blue (HID-AB) staining, and immunohistochemistry for olfactory marker protein (OMP), βIII tubulin (βIIIT), and Ki67. The lesions in the neuroepithelium and Bowman’s glands were quantitatively assessed by morphometric analyses of sections stained with anti-OMP antibody or HID-AB. The first appearance of neuroepithelial abnormality was observed in the dorsomedial portion of the olfactory mucosa in 5-month-old mice. The distribution and severity of lesions progressed with increasing age. In mildly affected epithelium in which OMP-positive olfactory receptor neurons (ORNs) were present but in smaller amounts, the numbers of βIIIT-positive and Ki67-positive neuroepithelial cells tended to be increased, indicating that neurogenesis was upregulated in these areas. In contrast, severely affected epithelium in which OMP-positive ORNs were virtually absent showed high variability in the numbers of βIIIT- and Ki67-positive cells among the areas examined, probably reflecting differences in the capacity of the basal cells remaining in the affected area to generate new neuronal cells. Histological analysis with HID-AB revealed that spontaneous lesions in Bowman’s glands also occurred in aged mouse olfactory mucosa. Lesions in the neuroepithelium and underlying Bowman’s glands tended to be spatially co-localized, suggesting a close association between pathogeneses in these two structures. Moreover, lesions in Bowman’s glands were associated with changes in the biochemical composition of mucus on the olfactory mucosa. This information should prove useful in improving the understanding of the pathogenetic mechanisms underlying age-related changes in the peripheral olfactory system. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (nos. 14770886, 16790987 and 18799002; K. Kondo) and a grant from the Japanese Ministry of Health, Labour, and Welfare (Comprehensive Research on Aging and Welfare, no. H13-choju-012; K. Nibu).     

Subcutaneous metastasis from transitional cell carcinoma of the bladder diagnosed by fine needle aspiration biopsy. A case report

BACKGROUND: Metastasis of transitional cell carcinoma (TCC) of the bladder to the skin and subcutaneous tissue is an uncommon finding. CASE: A 58-year-old man with a known case of high grade TCC of the bladder, presented with a right paraspinal mass. Clinically an abscess was suspected. Fine needle aspiration (FNA) showed many clusters and isolated malignant cells in an inflammatory background. The smears were diagnosed as positive for malignancy. CONCLUSION: It is essential to differentiate tumors metastatic to the skin and subcutaneous tissue from inflammatory lesions. FNA helped with the diagnosis in this case and prevented unnecessary biopsy.     

Loss of LETS protein is not a marker for salivary gland or bladder epithelial cell transformation

LETS protein was demonstrated by indirect immunofluorescence techniques in adult mouse submandibular gland and bladder epithelial cells at various stages during neoplastic transformation, after the application of a chemical carcinogen in vitro. There was no clear relationship between transformation and the loss of LETS protein; most normal and preneoplastic epithelium lacked the distinctive fibrillar distribution of fluorescent staining. In contrast, some carcinoma-producing cell lines did possess appreciable amounts of LETS protein.