Expression of keratins during experimentally induced carcinogenesis in hamster cheek pouch visualized polyclonal and monoclonal antibodies

Summary We obtained immnohistochemical profiles of several keratin proteins during experimentally induced carcinogenesis in hamster cheek-pouch mucosa using a polyclonal antibody (TK; detecting keratins with molecular masses of 41 65 kilodalton) and two monoclonal antibodies (KL1, 55- to 57-kilodalton keratins; PKK1; 40-, 45- and 52.5-kilodalton keratins). The squamous epithelium of normal pouch mucosa exhibited positive TK staining in all layers. KL1 staining in the spinous layer and PKK1 staining in the basal layer, thus indicating a regional or zonal distribution pattern. Epithelia undergoing basal hyperplasia showed irregular localization of PKK1 binding, while hyperkeratinized lesions exhibited the binding pattern found in normal epithelium. In case of epithelial dysplasia, there was reduced KL1 staining in spinous cells and decreased PKK1 staining in the basal and parabasal layers. Papillomas exhibited a rather zonal distribution of keratin staining. All squamous-cell carcinomas, irrespective of their degree of keratinization and infiltration pattern, showed slight or no PKK1 staining. Such lesions were only positive for KL1-detectable keratins in keratinizing tumour cells and exhibited an irregular distribution of TK binding. The expression of keratin proteins during carcinogenesis in hamster cheekpouch mucosa may parallel that of keratins in human squamous-cell carcinomas originating in the oral mucosa.     

Expression of keratins during experimentally induced carcinogenesis in hamster cheek pouch visualized polyclonal and monoclonal antibodies

We obtained immunohistochemical profiles of several keratin proteins during experimentally induced carcinogenesis in hamster cheek-pouch mucosa using a polyclonal antibody (TK; detecting keratins with molecular masses of 41-65 kilodalton) and two monoclonal antibodies (KL1, 55- to 57-kilodalton keratins; PKK1; 40-, 45- and 52.5-kilodalton keratins). The squamous epithelium of normal pouch mucosa exhibited positive TK staining in all layers, KL1 staining in the spinous layer and PKK1 staining in the basal layer, thus indicating a regional or zonal distribution pattern. Epithelia undergoing basal hyperplasia showed irregular localization of PKK1 binding, while hyperkeratinized lesions exhibited the binding pattern found in normal epithelium. In case of epithelial dysplasia, there was reduced KL1 staining in spinous cells and decreased PKK1 staining in the basal and parabasal layers. Papillomas exhibited a rather zonal distribution of keratin staining. All squamous-cell carcinomas, irrespective of their degree of keratinization and infiltration pattern, showed slight or no PKK1 staining. Such lesions were only positive for KL1-detectable keratins in keratinizing tumour cells and exhibited an irregular distribution of TK binding. The expression of keratin proteins during carcinogenesis in hamster cheek-pouch mucosa may parallel that of keratins in human squamous-cell carcinomas originating in the oral mucosa.     

Effect of epidermal chalones on the development and growth of cheek pouch tumors in the hamster

To the cheek pouch mucosa of 118 male Syrian golden hamsters 0.5% acetone solution of 7,12-dimethylbenz (a) anthracene was applied 3 times a week during 2 months. Two months after beginning of the experiments all the hamsters developed tumors. Since that time a mixture of epidermal G1 and G2 chalones was applied to the cheek pouch mucosa of the experimental animals 5 times a week. The control hamsters received saline alone. As compared to the control, the experimental hamsters demonstrated an increase in the life-span, tumor growth retardation and regression of part of the tumors. The data obtained attest to the anti-neoplastic action of chalones.     

Bradykinin- and substance P-induced edema formation in the hamster cheek pouch is tyrosine kinase dependent.

The purpose of this study was to determine whether protein tyrosine kinase, a ubiquitous family of intracellular signaling enzymes that regulates endothelial cell function, modulates bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. Using intravital microscopy, I found that suffusion of bradykinin or substance P (each, 0.5 and 1.0 microM) onto the cheek pouch elicited significant, concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa; P < 0.05). These responses were significantly attenuated by suffusion of genistein (1.0 microM) or tyrphostin 25 (10 microM), two structurally unrelated, nonspecific protein tyrosine kinase inhibitors (P < 0.05). Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P. Both inhibitors had no significant effects on adenosine (0.5 M)-induced responses (P > 0.5). Collectively, these data suggest that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion.     

The circadian variations in the epithelial growth of the hamster cheek pouch: quantitative analysis of DNA distributions

The pronounced diurnal rhythm in DNA distributions of the hamster cheek pouch epithelium both in the S fraction and in the (G2 + M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. The DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. The DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. The S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. The DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2 + M) phase and returning to G1 phase within a 24 hr period.     

Evaluation of chemopreventive effects of Thymoquinone on cell surface glycoconjugates and cytokeratin expression during DMBA induced hamster buccal pouch carcinogenesis

The present study aimed to investigate the membrane stabilizing effect of Thymoquinone (TQ) on cell surface glycoconjugates and cytokeratin expression against DMBA induced hamster buccal pouch carcinogenesis. 0.5% DMBA painting (three times per week) in hamster buccal pouches for 14 weeks resulted in the formation of well developed oral squamous cell carcinoma. We observed 100% tumor formation with marked abnormalities of glycoconjugates status in tumor bearing hamsters as compared to control animals. Oral administration of TQ at a dose of 30 mg/kg body weight, to DMBA painted hamsters on alternate days for 14 weeks, reduced the tumor formation as well as protected the levels of cell surface glycoconjugates in DMBA painted hamsters. The present study thus suggests that TQ has potent chemopreventive efficacy as well as protected the abnormalities on cell surface glycoconjugates during DMBA induced hamster buccal pouch carcinogenesis.     

Muscle fiber type is associated with obesity and weight loss

The purpose of this study was to test the hypothesis that muscle fiber type is related to obesity. Fiber type was compared 1) in lean and obese women, 2) in Caucasian (C) and African-American (AA) women, and 3) in obese individuals who lost weight after gastric bypass surgery. When lean (body mass index 24.0 +/- 0.9 kg/m(2), n = 28) and obese (34.8 +/- 0.9 kg/m(2), n = 25) women were compared, there were significant (P < 0.05) differences in muscle fiber type. The obese women possessed fewer type I (41.5 +/- 1.8 vs. 54.6 +/- 1.8%) and more type IIb (25.1 +/- 1.5 vs. 14.4 +/- 1.5%) fibers than the lean women. When ethnicity was accounted for, the percentage of type IIb fibers in obese AA was significantly higher than in obese C (31.0 +/- 2.4% vs. 19.2 +/- 1.9%); fewer type I fibers were also found in obese AA (34.5 +/- 2.8% vs. 48.6 +/- 2.2%). These data are consistent with the higher incidence of obesity and greater weight gain reported in AA women. With weight loss intervention, there was a positive relationship (r = 0.72, P < 0.005) between the percentage of excess weight loss and the percentage of type I fibers in morbidly obese patients. These findings indicate that there is a relationship between muscle fiber type and obesity.     

Effect of freezing and thawing on the microcirculation and capillary endothelium of the hamster cheek pouch

Golden Syrian hamster cheek pouches were prepared in vivo for observation of microvasculature under the light microscope. On an apparatus designed especially for this purpose, pouches were cooled from plus 10 °C to minus 30 °C at 1 °C/min, held at minus 30 °C for 1 min and warmed at 1 °C/min to plus 10 °C whence spontaneous rewarming to ambient was permitted. Pouches were observed under the light microscope throughout freezing and for 1 hr after thaw. At 0, 1, 5, 15, 30, and 60 min after thaw pouch tissue was taken for electron microscopy. After thaw hemostasis evolved in a biphasic pattern. In the early phase in which the degree of stasis reached its peak at about 20 min after thaw, cessation of circulation was caused by obstructive embolic platelet aggregates and a second uncharacterized factor, possibly humoral in nature. After 30 min, blood flow spontaneously resumed in at least one portion of each pouch. In 6 specimens, by 45–50 min, blood flow had returned to normal in about 90% of the microvasculature. After 50 min hemostasis began again and by 60 min blood flow had ceased in virtually all areas of the pouch, this time in the absence of vascular obstruction by platelet aggregates. Ultrastructural damage to endothelial cells occurring immediately at thaw and progressing through 1 hr thereafter included the rupture of cell membranes, early thinning, and later condensation of ground substance, decrease in the number and concentration of pinocytotic vesicles, swelling of rough endoplasmic reticulum, and the swelling and loss of cristal structure of mitochondria. This endothelial destruction can account for the permanent hemostasis of frostbite.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2 in vitro

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5-15 nmole/10 microliters) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI2. Three drugs were tested in this model. Cyclooxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formation of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogenous PGI2, since aggregation of hamster platelets by ADP was not inhibited by indomethacin in vitro. EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.), which has been proven to enhance PGI2 biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombotic drugs.     

Osteopontin is associated with nuclear factor kappaB gene expression during tail-suspension-induced bone loss

Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN-/- mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN-/- mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-kappaB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN-/- mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.     

Regulation of flow and wall shear stress in arteriolar networks of the hamster cheek pouch.

Our purpose was to define arteriolar network hemodynamics during moderate increases in interstitial adenosine or nitric oxide in the hamster (n = 34, pentobarbital sodium 70 mg/kg) cheek pouch tissue. The network consists of a feed arteriole (approximately 12-microm diameter, approximately 800-microm length) with three to six branches. Observations of diameter, red blood cell flux, and velocity were obtained at the feed before the branch and within the branch. A comparison of baseline with suffused adenosine or sodium nitroprusside (SNP) 10(-9) to 10(-5) M showed the following. First, diameter change was heterogeneous by agonist, did not reflect the expected dilatory response, and was related to location within the network. With adenosine, upstream branch points constricted and those downstream dilated, even at 10(-5) M. With SNP, upstream branch points dilated, whereas those downstream constricted. Second, with adenosine, changes in diameter, flux, and velocity together resulted in no change in wall shear stress until 10(-5) M. Wall shear stress was not maintained at a constant level with Nomega-nitro-L-arginine (10(-5) M), suggesting a role for flow-dependent diameter changes with adenosine. With SNP, diameter change correlated with the baseline (before SNP) shear stress conditions.     

Induction of proliferation by high molecular weight B cell growth factor or low molecular weight B cell growth factor is associated with increases in intracellular calcium in different subpopulations of human B lymphocytes

The activation of resting B cells with anti-surface Ig is associated with transient increases in intracellular calcium. In the present study, we demonstrate that stimulation of B cells which have already been activated by Staphylococcus aureus Cowan I (Sac), with high molecular weight B cell growth factor (HMW-BCGF) or low molecular weight B cell growth factor (LMW-BCGF), but not IL-2, IL-4, or interferon-gamma, is associated with an increase in intracellular calcium, which is modest compared to that seen with anti-Ig (approximately 100 nM vs approximately 400 nM). The increases in intracellular calcium induced by HMW-BCGF or LMW-BCGF occur in distinct but overlapping subpopulations of B cells. Thus, increases in intracellular calcium in human B cells occur not only upon activation but also upon the induction of proliferation by certain (but not all) B cell growth factors. Presumably, the effect of increasing intracellular calcium during the induction of proliferation is to modify a different group of intracellular molecules than those induced during activation.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5–15 nmol/10 μl) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI₂. Three drugs were tested in this model. Cycloxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formatiion of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogemous PGI₂, since aggregation of hamster platelets by ADP was not inhibited by indomethacin . EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.) which has been proven to enhance PGI₂ biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombic drugs.     

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.