Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction.

Contractile epithelial tubes are found in various organs, such as lung airways and blood capillaries. Their ability to sense luminal pressure and respond with adequate contractility is essential for their physiology, and its mis-regulation results in diseases such as asthma and hypertension. Here, we describe a mechanoresponsive regulatory pathway downstream of tissue stretching that controls contraction of the C. elegans spermatheca, a tubular structure where fertilization occurs. Using live-imaging, we show that ovulation-induced stretching of spermathecal cells leads to recruitment of the RhoGEF RHGF-1 to stress fibers, which activates RHO-1 and myosin II in a positive feedback loop. Through deletion analysis, we identified the PDZ domain of RHGF-1 as responsible for F-actin binding, and genetic epistasis analysis with the RhoGAP spv-1 demonstrated that tension-dependent recruitment of RHGF-1 to F-actin is required for robust spermathecal contractility. Our study illustrates how mechanosensitive regulators of Rho GTPases provide epithelial tubes the ability to tune their contractility in response to internal pressure.

Functional Insights into Protein Kinase A (PKA) Signaling from C. elegans.

Protein kinase A (PKA), which regulates a diverse set of biological functions downstream of cyclic AMP (cAMP), is a tetramer consisting of two catalytic subunits (PKA-C) and two regulatory subunits (PKA-R). When cAMP binds the PKA-R subunits, the PKA-C subunits are released and interact with downstream effectors. In Caenorhabditis elegans (C. elegans), PKA-C and PKA-R are encoded by kin-1 and kin-2, respectively. This review focuses on the contributions of work in C. elegans to our understanding of the many roles of PKA, including contractility and oocyte maturation in the reproductive system, lipid metabolism, physiology, mitochondrial function and lifespan, and a wide variety of behaviors. C. elegans provides a powerful genetic platform for understanding how this kinase can regulate an astounding variety of physiological responses.

Sorafenib inhibits MAPK-mediated proliferation in a Barrett's esophageal adenocarcinoma cell line.

SUMMARY: Esophageal adenocarcinoma continues to rise in incidence. Despite recognition of Barrett's metaplasia as the histological precursor, prognosis remains poor. The mitogen-activated protein kinases (MAPK) pathway is activated in Barrett's-associated dysplasia and adenocarcinoma and this activation is, in part, due to acid and bile acid reflux. We investigated the effects of sorafenib, an orally active Raf-inhibitor, on acid and bile acid-stimulated growth and signaling in SEG-1 cells, derived from a Barrett's esophageal cancer. SEG-1 cells were pretreated with sorafenib or vehicle and subsequently stimulated with acid or bile acid. MAPK signals, including phospho-ERK and phospho-p38, as well as cyclin D1 expression were assessed by Western blotting. Cell proliferation was measured by WST-1 colorimetric assay. Acid (pH 3.0-4.0) and bile acid (taurocholate 50-100 micromol/L) activated ERK and p38. Acid and bile acid exposure also increased levels of cyclin D1, a G1 to S cell cycle regulator. Furthermore, acid and taurocholate exposure increased cell proliferation. Sorafenib abrogated MAPK activation and cyclin D1 up-regulation and significantly inhibited cell growth. In summary, sorafenib inhibits acid or bile acid-stimulated Barrett's esophageal cancer cell proliferation by a mechanism involving the MAPK pathway. Our results suggest that sorafenib might be useful in the management of Barrett's-associated dysplasia and adenocarcinoma. These findings provide a foundation for in vivo studies to assess the efficacy of sorafenib in Barrett's-related neoplasia.

Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.

PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-zeta was not changed by PKC-delta siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-delta transfectants, compared to EV cells, PKC-delta upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-delta specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-delta regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-delta that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-delta in colonic carcinogenesis.

PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells.

BACKGROUND & AIMS: Previous studies showed decreased protein kinase C (PKC)-delta expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-delta on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells. METHODS: Human PKC-delta complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-delta transfectants were characterized by Western blotting. PKC-delta activity was measured in situ using a PKC-delta-specific substrate. Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry. In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining. RESULTS: In the presence of Zn(2+), PKC-delta transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-delta. PKC-delta transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype. Increased PKC-delta expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05). CONCLUSIONS: Our studies show that increased expression of PKC-delta inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.

Inhibition of O(6)-methylguanine-DNA methyltransferase increases azoxymethane-induced colonic tumors in rats.

Azoxymethane (AOM) causes O(6)-methylguanine adduct formation which leads to G-->A transitions. Their repair is carried out by O(6)-methylguanine-DNA methyltransferase (MGMT). To evaluate the importance of this repair event in AOM-induced carcinogenesis, we examined the effect of O(6)-benzylguanine (BG), a potent inhibitor of MGMT, on colonic tumor development. Rats were treated weekly for 2 weeks at 0 and 24 h with BG (60 mg/kg body wt i.p.) or vehicle (40% polyethylene glycol, PEG-400), followed 2 h after the first dose of BG with AOM (15 mg/kg body wt) or vehicle (saline) i.p. Rats were killed 35 weeks later and tumors harvested and DNA extracted. In the AOM-treated groups, BG caused a significant increase in tumor incidence with tumors in 65.9%, versus 30.8% in the AOM/PEG-treated group (P < 0.05). In the BG/AOM group there was also a significant increase in tumor multiplicity, with 2.3 tumors/tumor-bearing rat, versus 1.6 tumors/tumor- bearing rat in the AOM/PEG group (P < 0.05). Since O(6)-methylguanine adducts can cause activating mutations in the K-ras and beta-catenin genes, we examined the effects of BG on these mutations. In the BG group there were seven mutations in codon 12 or 13 of exon 1 of the K-ras gene in 51 tumors examined, compared with no K-ras mutations in 17 tumors analyzed in the AOM/PEG group (P = 0.12). In the BG/AOM group there were 10 mutations in exon 3 of the beta-catenin gene among 48 tumors evaluated, compared with six mutations in 16 tumors analyzed in the PEG/AOM group (P = 0.16). In summary, MGMT inhibition increases AOM-induced colonic tumor incidence and multiplicity in rats.

DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells.

The effects of the Auger electron-emitting isotope 123I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the 123I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with 60Co gamma rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the 123I-labeled estrogen. The 123I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of 123I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that 123I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 +/- 1.7, which is similar to that seen with high-LET radiations. Our results suggest that 123I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by 123I bound to estrogen is very like damage induced by high-LET radiation, and the 123I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA.

DNA double-strand break rejoining rates, inherent radiation sensitivity and human tumour response to radiotherapy.

The relationship between DNA double-strand break rejoining rates, inherent radiation sensitivity and tumour response to radiation therapy was determined for a group of 25 squamous cell carcinoma (SCC) and eight sarcoma (SAR) tumours. DNA double-strand break frequencies were measured by neutral filter elution in first passage following explant tumour samples after in vitro exposure to 100 Gy of 60Co gamma-rays. There was no significant difference between SCC and SAR tumour cells in their sensitivity to break induction, but in a 1 h time period SAR tumour cells rejoined significantly fewer breaks than SCC tumour cells, consistent with the greater sensitivity of SAR and suggesting that differences in rates of break rejoining account for the different distributions of radiosensitivities seen when different tumour types are compared. The percentage of breaks rejoined in 1 h in these tumour samples correlated well with D(o) and with the beta component of the survival curve, measured in vitro by clonogenic assay in tumour cell lines established from the tumour samples, but not with SF2 or the alpha component of the survival curve. The rates of DNA double-strand break rejoining therefore appear to influence the exponential portion of survival curves and probably the interactions between breaks. The percentage of breaks rejoined in 1 h was higher in SCC tumours that subsequently failed radiotherapy and, although the differences were not significant, they suggest that rates of break rejoining are an important component of tumour response to radiation therapy.

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Enhancement of X-ray toxicity in squamous cell carcinoma cell lines by DNA polymerase inhibitors.

The effect of the DNA polymerase inhibitors adenine 9-beta-arabinofuranoside (ara-A), cytosine 1-beta-arabinofuranoside (ara-C), and aphidicolin on X-radiation sensitivity was studied in a group of exponentially growing squamous cell carcinoma cell lines. The tumour cell lines varied in radiation sensitivity, with D0 (radiation sensitivity) values ranging from 1.0 to 3.9 Gy. The addition of non-toxic concentrations of ara-A 30 min before irradiation and removal 30 min after irradiation potentiated cell killing in five of eight cell lines. Four of these five responsive cell lines were relatively radioresistant lines, having D0 > 2.0 Gy. One of the cell lines was more radiosensitive (D0 = 1.4 Gy). Ara-A was also effective in potentiating killing in the radioresistant cell lines even when added 60 min after irradiation. Pre- or post-treatment with ara-A had no effect on X-ray sensitivity of the other three relatively sensitive cell lines (D0 ranging from 1.0 to 1.3 Gy). Both ara-C and aphidicolin were effective in potentiating X-ray sensitivity in JSQ-3, a relatively resistant cell line that was sensitized by ara-A treatment, but they had no effect on the X-ray sensitivity of SCC-61, a relatively radiosensitive cell line that was insensitive to ara-A effects on X-ray response. At the concentrations used, the polymerase inhibitors were equally effective in inhibiting DNA synthesis.

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.

Inhibition of protein kinases sensitizes human tumor cells to ionizing radiation.

Protein kinase C (PKC) is activated rapidly and transiently following ionizing radiation exposure and is postulated to activate downstream nuclear signal transducers. Inhibition of this enzyme attenuates radiation-mediated expression of the c-jun and Egr-1/zif-268 genes which are associated with cellular proliferation. To investigate further the role of PKC in the radiation response of human tumor cell lines, two human squamous cell carcinoma cell lines, SQ-20B and JSQ-3, were exposed to graded doses of X rays in the presence of staurosporine, sangivamycin, or H7, all PKC inhibitors. The protein kinase inhibitors staurosporine and sangivamycin produced dose-dependent cytotoxicity in cells of the SQ-20B and JSQ-3 cell lines while H7 did not. Nontoxic concentrations of sangivamycin (10 nM) and staurosporine (1 nM), added to cell cultures from 1 to 7 h before X irradiation, enhanced cell killing by radiation in both cell lines. Maximal sensitization of killing occurred when inhibitors were added 1 h prior to irradiation. The enhanced radiation-induced cell killing was not due to any measurable alteration in the induction or rejoining of DNA single- or double-strand breaks as determined by alkaline and neutral filter elution assays. These data suggest that protein kinase activity is important for cell survival following radiation exposure, although the specific role of PKC in radiation responses is unknown.

Radiation-induced DNA double-strand break frequencies in human squamous cell carcinoma cell lines of different radiation sensitivities.

DNA neutral (pH 9.6) filter elution was used to measure radiation-induced DNA double-strand break (dsb) frequencies in eight human squamous cell carcinoma cell lines with radiosensitivities (D0) ranging from 1.07 to 2.66 Gy and D values ranging from 1.46 to 4.08 Gy. The elution profiles of unirradiated samples from more radiosensitive cell lines were all steeper in slope than the profiles from resistant cells. The shapes of the dsb induction curves were curvilinear and there was some variability from cell line to cell line in the dose-response for the induction of DNA dsb after exposures to 5-100 Gy 60Co gamma-rays. There was no relation between the shapes of the survival curves and the shapes of the dose-responses for the induction of DNA dsb. At low doses (5-25 Gy), three out of four of the more sensitive cell lines (D less than 2.5 Gy) had larger initial break frequencies than the more resistant lines (D greater than 3.0 Gy). Although the low-dose (5-25 Gy) elution results were variable, they do suggest that DNA neutral elution will detect differences between sensitive and resistant tumour cells in initial DNA dsb frequencies.

Prediction of the radiation sensitivity of human squamous cell carcinoma cells using DNA filter elution.

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.