Modulating influence of dehydroepiandrosterone administration on the morphology and enzyme phenotype of dimethylaminoazobenzene-induced hepatocellular foci and nodules

The effect of 4 weeks dietary administration of the hormone dehydroepiandrosterone (DHEA) on enzyme and morphological phenotype of focal lesions previously induced by dimethylaminoazobenzene (DAB) treatment was investigated in Sprague-Dawley rats. In contrast to the DAB-alone livers where large numbers of glycogen-storing, mixed cell nodules homogeneously positive for glutathione S-transferase P form (GST-P) were apparent, DHEA treated animals were characterized by significantly fewer, more heterogeneous lesions, in some cases demonstrating increased amphophilia and structured basophilia. The enhanced heterogeneity, in some ways reminiscent of that reported earlier for ‘reversibility’ or ‘remodelling’ of rapidly induced nodular lesions, was associated with increased catalase (CAT), acid phosphatase (AP) and glucose-6-phosphatase (G6Pase) and decreased glycogen content and phosphorylase (PHO) activity in both nodules and background parenchyma. Glucose-6-phosphate dehydrogenase (G6PD) activity was elevated irregularly, focal lesions also demonstrating a heterogeneous reaction. The experimental data suggest two separate effects of the hormone treatment, the first involving modulation of the usual altered phenotype of preneoplastic lesions with a shift towards ‘tigroid’ cell character and the second, similar to that reported earlier for rapidly induced nodules, involving enhanced phenotypic instability and leading to reduction in numbers.     

Modulating influence of dehydroepiandrosterone administration on the morphology and enzyme phenotype of dimethylaminoazobenzene-induced hepatocellular foci and nodules

The effect o 4 weeks dietary administration of the hormone dehydroepiandrosterone (DHEA) on enzyme and morphological phenotype of focal lesions previously induced by dimethylaminoazobenzene (DAB) treatment was investigated in Sprague-Dawley rats. In contrast to the DAB-alone livers where large numbers of glycogen-storing, mixed cell nodules homogeneously positive for glutathione S-transferase P form (GST-P) were apparent, DHEA treated animals were characterized by significantly fewer, more heterogeneous lesions, in some cases demonstrating increased amphophilia and structured basophilia. The enhanced heterogeneity, in some ways reminiscent of that reported earlier for 'reversibility' or 'remodelling' of rapidly induced nodular lesions, was associated with increased catalase (CAT), acid phosphatase (AP) and glucose-6- phosphatase (G6Pase) and decreased glycogen contents and phosphorylase (PHO) activity in both nodules and background parenchyma. Glucose-6-phosphatase (G6PD) activity was elevated irregularly focal lesions also demonstrating a heterogeneous reaction. The experimental data suggest two separate effects of the hormone treatment the first involving modulation of the usual altered phenotype of preneoplastic lesions with a shift towards 'tigroid' cell character and the second, similar to that reported earlier for rapidly induced nodules, involving enhanced phenotypic instability and leading to reduction in numbers.     

Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.     

The evolving concept of tumor microenvironments

The role of the microenvironment in cancer development is being increasingly appreciated. This paper will review data that highlight an emerging distinction between two different entities: the microenvironment that altered/preneoplastic/neoplastic cells find in the tissue where they reside, and the peculiar microenvironment inside the focal lesion (tumor) that these cells contribute to create. While alteration in the tissue environment can contribute to the selective clonal expansion of altered cells to form focal proliferative lesions, the atypical, non-integrated growth pattern that defines such focal lesions leads to the appearance of what is correctly referred to as the tumor microenvironment. The latter represents a new and unique biological milieu, characterized by hypoxia, acidosis and other biochemical and metabolic alterations, including genetic instability, that can set the stage for tumor progression to occur. Thus, the two microenvironments act in sequence and play complementary roles in the development of overt neoplasia. This distinction has important implications for the understanding of disease pathogenesis and for the management of preneoplastic/neoplastic lesions at various stages of cancer development.     

Immunohistochemically demonstrated increase in glutathione S-transferase species in propylnitrosamine-induced focal proliferative and neoplastic Syrian hamster pancreatic lesions

Immunohistochemical investigation of focal proliferative and neoplastic Syrian hamster pancreatic lesions induced by propylnitrosamine administration revealed a distinct pattern of expression of different molecular forms of glutathione S-transferase (GST) during neoplastic development. Initial increase in levels of GST placental (P) and B forms in early ductal/ductular proliferations and atypical (dysplastic) lesions was followed by a drop in the latter during transition to carcinoma. Unequivocal acinar cells observed within so-called 'pseudoductules' did not share the altered phenotype evident in ductular elements, suggesting their non-involvement in the generation of early lesions. However, the fact that component cells were occasionally of abnormal morphology did not allow the exclusion of acinar cell participation in histogenesis. Elevation of GST-A and C forms was limited to stromal elements surrounding the epithelial lesions and since they were associated with benign, cystic as well as atypical lesions and a similar increase was observed after common duct ligation, they appeared to be non-specific. The results indicated independent control of expression of individual GST forms and suggest that biochemical similarities exist between early, putative preneoplastic lesions induced by propylnitrosamines in the hamster lung, liver (both hepatocellular and intrahepatic bile duct) and pancreas.     

Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus.

The proliferative disease systemic neoplasia, also termed hemic neoplasia or disseminated sarcoma, was studied in four Puget Sound, Washington populations of the bay mussel (Mytilus sp.). Using flow cytometric measurement of DAPI-stained cells withdrawn from the hemolymph, DNA content frequency histograms were generated for 73 individuals affected by the disease. The cells manifesting systemic neoplasia were found to exist as either of two separate types, characterized by G0G1 phase nuclear DNA contents of either approximately 4.9 x haploid (pentaploid form) or approximately 3.8 x haploid (tetraploid form). The two disease forms were found to coexist in all four mussel populations sampled, with overall relative prevalences of 66% pentaploid form, 29% tetraploid form, and 5% exhibiting both disease forms simultaneously. These findings represent the first unequivocal demonstration of multiple cell types in a bivalve neoplasia. The two forms appear to represent separate pathogenetic processes rather than sequential stages of a single pathogenesis. Two cell cycling parameters associated with proliferative activity were employed to compare the alternate forms: (i) the percentage of cells assigned to the DNA Synthesis (S) phase of the neoplastic cell cycle, and (ii) the proportion of neoplastic cell mitotic figures in hemocytological preparations. Mean values for both parameters were significantly higher for mussels with the tetraploid form of the disease, suggesting a higher rate of proliferation relative to the pentaploid form. Qualitatively, cells of the tetraploid form contained slightly lower nuclear and cytoplasmic volumes compared to those of the pentaploid form. An observed wide variation in neoplastic cell nuclear size within either disease form may reflect the distribution of cells in the G0G1, S, and G2M phases of the cell cycle. Potential etiologic relationships between the two forms are discussed.     

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.     

Intestinal metaplasia and altered enzyme expression in propylnitrosamine-induced Syrian hamster cholangiocellular and gallbladder lesions

Intrahepatic bile duct and gallbladder preneoplastic and neoplastic lesions induced in Syrian golden hamsters by propylnitrosamine treatment were investigated for the presence of polysaccharides and assayed immunohistochemically for expression of the enzymes glucose-6-phosphate dehydrogenase (G6PD) and glutathione-S-transferase (GST) molecular forms. On the basis of an increase in G6PD and the GST-placental form, a sequence of altered cell populations ranging from simple ductular proliferation through dysplasia and cholangiofibroma to cholangiocellular carcinoma could be established, the latter three lesions being characterized by marked increase in polysaccharide production. While similar goblet cell (intestinal) metaplasia and increased polysaccharide storage were also evident in carcinogen-induced gallbladder lesions G6PD and GST-P expression was decreased when compared with control epithelium.     

Cytometric evidence that cervical intraepithelial neoplasia I and II are dysplasias rather than true neoplasias. An image analysis study of factors involved in the progression of cervical lesions.

Image analysis was performed on 40 Feulgen-stained histologic samples and 48 Feulgen-stained cytologic preparations representing normal squamous epithelium and all grades of cervical lesions (from mild dysplasia to invasive carcinoma) in order to characterize the evolutionary progressive changes in cervical epithelial proliferative disease toward malignancy. Quantitative studies included the analysis of proliferative features, differentiation features, nuclear morphology and DNA content. The data obtained on the histologic sections showed that the various features, to a different extent, detected a gradual increase in phenotypic cellular disarrangements related to the progression of the cervical lesions toward malignancy--that is, the modifications to nuclear area, perimeter, DNA content, percentage of nuclei with nucleoli, nuclear/cytoplasmic ratio and percentage of cells with no membrane positivity for soybean agglutinin lectin were progressively greater, moving from normal epithelium and mild dysplasia toward infiltrating carcinoma. In particular, all the morphologic and histochemical features appeared to parallel a diploid reduction and the appearance of aneuploidy. The simultaneous evaluation of proliferation- and differentiation-related features, together with those of nuclear DNA content, showed two main successive preneoplastic lesions: one characterized by an increase in cell turnover without alterations in its organization and another by a true neoplastic disorder. The data obtained on sequential cytologic examinations showed that individual cell changes are detectable and seem basically to be characterized by the appearance of clusters of cells with somatic characteristics not observed in previous cytologic checks. From the results of our study, the cervical intraepithelial neoplasia (CIN) concept appears to be inaccurate. In fact, only CIN III (severe dysplasia/carcinoma in situ) lesions have the morphologic and proliferative alterations of true neoplasia. In contrast, CIN I and some cases of CIN II lesions lack these characteristics and seem to be properly classified as dysplasia, thus avoiding the term neoplasia, implicit in CIN. Moreover, the multivariate study of data sets of features related to the progressive somatic changes, both in histologically and cytologically studied cases, allows us to detect the steps of progression; they are marked by the appearance of cell clusters with qualitatively different phenotypic characters when compared to the cell populations from which they presumably arise. These results seem to provide a further argument against the CIN theory, which stresses the concept that progression is related only to a gradual numerical increase in an initially established phenotype with the characteristics of malignancy.     

Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.     

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.     

Iron exclusion by proliferative foci and neoplastic lesions induced by 7,12-dimethylbenz (a) anthracene in the rat adrenal cortex

Summary Proliferative and neoplastic adrenal cortical lesions were produced in female Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene. These lesions were studied for their ability to store histochemically demonstrable cellular iron compared with normal adrenocortical cells following iron loading by subcutaneous injection of iron-dextran. Stainable iron was excluded, under conditions in which normal cortical cells were heavily loaded with iron, in three histological types of adrenalcortical lesions; eosinophilic and basophilic proliferative foci; adenomas and lesions transitional between foci and tumours. The property of iron exclusion may be useful in studying neoplastic development in the adrenal gland.     

A case of ataxia telangiectasia with unbalanced glucose 6-phosphate dehydrogenase mosaicism in the granulocytic/monocytic lineages.

Ataxia telangiectasia is a genetically determined disease with multi-system abnormalities and a high incidence of neoplasia. In order to define the nature of the association between ataxia telangiectasia and malignancy, we investigated a patient with the disease and heterozygote for the Mediterranean variant of the X-linked marker glucose 6-phosphate dehydrogenase. Enzymatic mosaicism in hemopoietic and nonhemopoietic cells was evaluated with the 2-deoxy glucose 6-phosphate technique. While erythrocytes, platelets, and lymphocytes expressed the same double-enzyme phenotype as tissues of nonhemopoietic origin, granulocytes and monocytes expressed almost exclusively the Mediterranean-type enzyme. We suggest that, as the result of genetic instability at the hemopoietic stem-cell level, the granulocytic/monocytic progeny enjoyed a proliferative advantage and became the predominant clone.     

Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells.

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.     

Atypical glandular cells: criteria to discriminate benign from neoplastic lesions and squamous from glandular neoplasia

OBJECTIVE: To evaluate the presence of some criteria in cervical smears with atypical glandular cells and their correlation with histological patterns to identify pre-neoplastic and neoplastic lesions. METHODS: Seventy-three women referred with an atypical glandular cell smear, who had undergone conization or hysterectomy, were included in this study. Referral Pap smears were reviewed using the set of 27 cyto-morphological criteria that was correlated with the histological diagnosis. RESULTS: Histological results showed intraepithelial or invasive neoplasia in 35 (48%) cases and benign lesions in 38 (52%) cases. After logistic regression and decision tree analysis an increased nuclear/cytoplasmic ratio and the presence of dyskeratotic cells were strongly associated with intraepithelial or invasive neoplasia and the differential cyto-morphological criteria for glandular lesions were decreased cytoplasm, irregular nuclear membranes and the presence of nucleoli. CONCLUSION: The analysis of individual cyto-morphological criteria can better predict intraepithelial or invasive neoplasia and differentiate glandular from squamous lesions.