Molecular genetics and cellular events of K-Ras-driven tumorigenesis.

Cellular transformation and the accumulation of genomic instability are the two key events required for tumorigenesis. K-Ras (Kirsten-rat sarcoma viral oncogene homolog) is a prominent oncogene that has been proven to drive tumorigenesis. K-Ras also modulates numerous genetic regulatory mechanisms and forms a large tumorigenesis network. In this review, we track the genetic aspects of K-Ras signaling networks and assemble the sequence of cellular events that constitute the tumorigenesis process, such as regulation of K-Ras expression (which is influenced by miRNA, small nucleolar RNA and lncRNA), activation of K-Ras (mutations), generation of reactive oxygen species (ROS), induction of DNA damage and apoptosis, induction of DNA damage repair pathways and ROS detoxification systems, cellular transformation after apoptosis by the blebbishield emergency program and the accumulation of genomic/chromosomal instability that leads to tumorigenesis.

Matrix metalloproteinases (mmp) in restorative dentistry and endodontics.

Matrix metalloproteinases (MMPs) seem to play a dual role in dentistry. While several MMPs have an important role to play in developmental defects of teeth and in caries, some MMPs also seem to have a defensive role. The main organic component of tooth structure is collagen and MMPs that degrade collagen and the extra cellular matrix have been implicated in progression of dental caries. MMPs have also been shown to be active in pulpitis and studies have shown that they can be used as diagnostic markers of pulpal inflammation. This paper reviews the role of MMPs in restorative dentistry and endodontics.

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes.

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.

Dynamics of E-cadherin and gamma-catenin complexes during dedifferentiation of polarized MDCK cells.

BACKGROUND: E-cadherin mediated cell-cell adhesion and hepatocyte growth factor (HGF) are important for renal epithelial morphogenesis. We previously showed that HGF dedifferentiates previously well polarized Madin-Darby canine kidney (MDCK) cell monolayers grown on filters. The regulation of E-cadherin during epithelial dedifferentiation is not known. We hypothesized that E-cadherin mediated cell-cell adhesion is modulated during HGF induced dedifferentiation of MDCK cell monolayers. METHODS: We analyzed E-cadherin/gamma-catenin interaction and distribution during epithelial dedifferentiation in vitro using a model of polarized MDCK cell monolayers treated with HGF. RESULTS: Surface immunoprecipitation experiments showed that HGF increased the amount of cell surface E-cadherin associated with gamma-catenin. Biochemical and morphological examination of the TX-100 solubility of junctional E-cadherin and gamma-catenin in control and HGF treated cells showed an increase in solubility of only E-cadherin during loss of cell polarity. Metabolic labeling of control and HGF treated cells showed that HGF stimulated the synthetic rate of E-cadherin and gamma-catenin molecules. Inulin flux across MDCK cell monolayers increases with HGF treatment. CONCLUSION: These data provide evidence for both the dissociation of E-cadherin molecules from the actin cytoskeleton and an increase in the total number of E-cadherin/gamma-catenin complexes on the cell surface during HGF-induced dedifferentiation of polarized renal epithelium. These data support the hypothesis that E-cadherin function is inhibited by a mechanism of detachment from the actin based cytoskeleton during HGF induced dedifferentiation of polarized renal epithelia.

Demonstration of the presence of cyclic inositol phosphohydrolase in human urine.

Cyclic inositol phosphohydrolase (cIPH), cleaves the cyclic bond of cyclic inositol monophosphate (cIP) to yield inositol monophosphate. In this communication, we demonstrate the presence of cIPH in human urine. cIPH was measured in the 24-h urine samples of both male and female hospital patients. cIPH released per day ranged from 0 to 243 units in men (n = 16) and from 15 to 346 units in women (n = 18). Release of cIPH activity was not related to renal function as measured by creatinine clearance. HPLC ion-exchange chromatography or HPLC gel filtration of ammonium sulfate precipitate yielded a distinct cIPH peak with an apparent molecular weight of 40 kDa on gel filtration. This is the first demonstration of the presence of this enzyme in human urine. The large variation (over 20-fold) in the excretion of this protein suggests that it may have physiological and/or pathological significance.

Dissociation of cyclic inositol phosphohydrolase activity from annexin III.

Cyclic inositol phosphohydrolase is a phosphodiesterase that cleaves the cyclic bond of cyclic inositol monophosphate. In 1990, Ross et al. (Ross, T. S., Tait, J. F., and Majerus, P. W. (1990) Science 248, 605-607) purified this enzyme from human placenta and reported that cyclic inositol phosphohydrolase is identical to annexin III. Independent confirmation of this finding has not been provided. The relative distribution of annexin III and cyclic inositol phosphohydrolase activity in rat kidney and spleen indicated that annexin III can be dissociated from cyclic inositol phosphohydrolase activity. Rat spleen contains large quantities of annexin III, but has very little cyclic inositol phosphohydrolase activity. In contrast, rat kidney, one of the richest sources of cyclic inositol phosphohydrolase activity, possesses very little (immunohistochemistry) or no (Western blot) annexin III. Similar to cytosol of human placenta, cytosol of guinea pig kidney contains both annexin III and cyclic inositol phosphohydrolase. On SDS-gel electrophoresis, guinea pig kidney annexin III has a slightly different mobility than the human placental annexin III. Human placental annexin III co-migrates with cyclic inositol phosphohydrolase on ion exchange chromatography, while guinea pig kidney annexin III is clearly dissociated from cyclic inositol phosphohydrolase on ion exchange chromatography. Both guinea pig kidney annexin III and human placental annexin III pellet with the addition of calcium and centrifugation, while cyclic inositol phosphohydrolase activity in both of these tissues remains in the supernatant. Our studies clearly show that cyclic inositol phosphohydrolase and annexin III are two different proteins.

Decreased cyclic inositol phosphohydrolase activity in hamster renal tumors and human renal cell carcinomas.

Cyclic inositol phosphohydrolase (cIPH) (EC 3.1.4.36) hydrolyzes the cyclic phosphodiester bond of cyclic inositol monophosphate to yield inositol 1-phosphate. Renal tissue in various species has been shown to contain the highest activity of this enzyme. Cyclic inositol monophosphate, the substrate for this enzyme, has been implicated in the regulation of cell density. Thus, we determined whether activity of this enzyme is modulated in renal tumor. Samples of hamster renal tumors induced by diethylstilbestrol (DES) demonstrated 87% less cIPH activity than control hamster kidney. After 8 days of DES treatment of hamsters, a 28% decrease in the cIPH activity in kidney was observed. Interestingly, comparison of the cIPH activity in normal human kidney cortex, medulla, and renal cell carcinoma gave the following values (mean +/- SD), 10.6 +/- 4.6 (9), 6.7 +/- 4.6 (5), and 1.9 +/- 2.3 (8), respectively. Our study demonstrates that cIPH activity is reduced in hamster and human renal tumors.

Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes.

Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.