Restoration of the rat urothelium after cyclophosphamide treatment.

Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.     

Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli

When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and 'rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of 'stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity.     

Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.     

Correlation of membrane-bound and free ribosomes in normal rat liver, Zajdela hepatoma rat liver and ascite cells proper]

The content of membrane-bound ribosomes in normal rat liver cells is 3 times as high as compared to that of free ribosomes. (K=membrane-bound ribosome RNAs divided by free ribosome RNAs=3, the opposite effect being observed in case of ascites hepatoma cells. A considerable increase in the free ribosome fraction in the liver of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of membrane-bound ribosomes (K=0.6). Similar, but less-pronounced changes were observed in liver cells of control animals after 48-hour starvation (K=0.9), simulating the condition occurring during the last days of tumour animals' life. Thus, changes in the rativ of membrane-bound to free ribosomes in liver during the ascites tumour growth are probably specifics and are not only due to anorexia in Zajdela hepatoma animals.     

Unequal distribution of osmiophilic particles in the epidermal periplasmic space of upper and lower flanks of gravi-responding rye coleoptiles

In various studies, auxin (IAA)-induced coleoptile growth has been reported to be closely correlated with an increased occurrence of osmiophilic particles (OPs) at the inner surface of the outer, growth-limiting epidermal cell wall, indicating a possible function related to the mechanism of IAA-induced wall loosening. In order to test whether changes in cell elongation rates of upper and lower flanks (UFs, LFs, respectively) during graviresponsive growth are reflected in appropriate changes in the occurrence of OPs, rye (Secale cereale L.) coleoptiles either as segments or as part of intact seedlings, were gravitropically stimulated by positioning them horizontally for 2 h. Ultrastructural analyses within the UFs and LFs of the upward-bending coleoptiles revealed a distinct imbalance in the occurrence of OPs. The number of OPs per transverse epidermal cell section of the elongation-inhibited UF on average amounted to twice the number of OPs counted in epidermal cell sections of the faster-growing LF. As a hypothesis, the results lead us to suggest that OPs are involved in the mechanism of wall loosening and that temporary growth inhibition of epidermal cells of the UF during upward bending is mediated by inhibition of OP entry into the cell walls. Thereby, more OPs accumulate near the inner surface of the outer wall of epidermal cells of the UF compared with the LF.     

Progesterone Deficiency and Premature Labour

Plasma oestradiol 17β and progesterone levels in 11 patients admitted to hospital for threatened premature labour of unknown aetiology were compared with those of women at similar stages of gestation whose pregnancy was normal. Oestradiol levels in the study group were slightly higher than in the normal controls but their progesterone levels were significantly lower. This progesterone deficiency increased the oestradiol/progesterone ratio in the study group patients, and it increased still more as the progesterone withdrawal continued during premature labour.Since uterine activity during pregnancy is regulated by a balanced action of several factors a deficiency in progesterone, an opponent of uterine activity, creates a regulatory imbalance which, if uncorrected, provokes premature labour. An increase in uterine volume stimulates uterine activity, and the present study reinforced our previous conclusion that the uterine-volume/plasma-progesterone ratio is a more accurate measure of the state of regulatory balance than the progesterone level alone.The cause of the progesterone deficiency in these cases remains unexplained, but we suggest that placental growth and function are contributory factors. We are investigating ways of correcting the resulting imbalance in the regulatory mechanism.     

Identification of genes differentially expressed in benign prostatic hyperplasia.

Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial-stromal homeostasis in BPH. (J Histochem Cytochem 49:669-670, 2001)     

Regulation of cytosolic phospholipase A2 in rat endometrial stromal cells: the role of epidermal growth factor

The effect of epidermal growth factor on the levels of cytosolic phospholipase A2 mRNA and protein in cultured rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor increased the steady-state cytosolic phospholipase A2 mRNA and protein levels as demonstrated by Northern and Western blot analyses, respectively. Immunocytochemical analysis demonstrated an increase of cytosolic phospholipase A2 protein in most cells, as opposed to a small subpopulation of cells in culture. These results show that epidermal growth factor causes an increase in steady-state cytosolic phospholipase A2 mRNA and protein levels in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction. Epidermal growth factor receptor ligands may regulate cytosolic phospholipase A2 and thus prostaglandin production in the endometrial stromal cells during implantation.     

Dorsal skin responses to subchronic ultraviolet B (UVB)-irradiation in Wistar-derived hypotrichotic WBN/ILA-Ht rats

Dorsal skin responses to a subchronic UVB-irradiation (10kJ/m2/rat /day), were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats for up to 3 months. Hyperplasia of epidermal cells and hair follicle epithelial cells as well as parakeratosis developed at 1 month and progressed thereafter, resulting in a prominent epidermis thickening and formation of epidermal ingrowths projecting into the dermis. At the same time, the percentage of proliferating cell nuclear antigen (PCNA)-positive epidermal cells significantly increased after I month. In some portions of the hyperplastic epidermis, especially of the epidermal ingrowths, keratinocytes were somewhat pleomorphic and migrated into the dermis. In the upper dermis, edema with capillary congestion, mast cell infiltration and fibroblast proliferation developed at I month, and the intensity of edema and the number of dermal mast cells was most prominent at 3 months. Edema spread to the epidermis, resulting in intercellular edema and subsequent dissociation of epidermal cells. Degeneration of collagen fibers was also detected in the upper dermis, especially beneath the epidermis. In addition, although not significant because of a large individual difference, the serum IgE concentration, showed a tendency to increase after 2 months. The present study clarified the characteristics of the dorsal skin responses to a subchronic UVB-irradiation in rats.     

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes.

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.     

ppGpp-mediated regulation of DNA replication and cell division in Escherichia coli

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis [4], ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

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.     

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.     

Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.