Contralateral exploration and repair of occult inguinal hernias during laparoscopic inguinal hernia repair: systematic review and Markov decision process.

BACKGROUND: Contralateral clinically occult hernias are frequently noted at the time of laparoscopic unilateral inguinal hernia repair. There is no consensus on the role of contralateral exploration and repair. This systematic review assessed the safety and efficacy of operative repair of occult contralateral inguinal hernias found during unilateral repair. METHODS: PubMed, Embase, and the Cochrane Central Register of Controlled Trials were searched from inception to February 2020. Adults diagnosed with a unilateral inguinal hernia undergoing laparoscopic repair were included. The primary outcome was the incidence of occult contralateral hernias. Summative outcomes of operative and expectant management were reported along with development of a Markov decision process. RESULTS: Thirteen studies (1 randomized trial, 12 observational cohorts) with 5000 patients were included. The incidence of occult contralateral inguinal hernias was 14.6 (range 7.3-50.1) per cent. Among patients who underwent repair, 10.5 (4.3-17.0) per cent experienced a postoperative complication. Of patients managed expectantly, 29 per cent later required elective repair for symptoms. Mean follow-up was 36 (range 2-218) months. Using a Markov decision process, it was calculated that, for every 1000 patients undergoing unilateral inguinal hernia repair, contralateral exploration would identify 150 patients with an occult hernia. Repair would result in 15 patients developing a postoperative complication and 105 undergoing unnecessary repair. Alternatively, expectant management would result in 45 patients requiring subsequent repair. CONCLUSION: Contralateral repair is not warranted in patients with occult hernias diagnosed at the time of elective hernia repair. The evidence is largely based on observational studies at high risk of bias.

Importance of the physical exam: double-blind randomized controlled trial of radiologic interpretation of ventral hernias after selective clinical information.

PURPOSE: Increasingly, radiologic imaging is obtained as part of the pathway in diagnosing ventral hernias. Often, radiologists receive incomplete or incorrect clinical information from clinicians. OBJECTIVE: The aim of the study is to determine if clinical exam findings alter radiological interpretation of ventral hernias on CT. METHODS: This is a single-institution double-blind, randomized trial. All patients with a recent abdominal/pelvic CT scan seen in various surgical clinics were enrolled. A surgeon blinded to the CT scan findings performed a standardized physical examination and assessed for the presence of a ventral hernia. Seven independent radiologists blinded to the study design reviewed the scans. Each radiologist received one of three types of clinical exam data per CT: accurate (correct), inaccurate (purposely incorrect), or none. Allocation was random and stratified by the presence of clinical hernia. The primary outcome was the proportion of radiologic hernias detected, analyzed by chi square. RESULTS: 115 patients were enrolled for a total of 805 CT scan reads. The proportion of hernias detected differed by up to 25% depending on if accurate, no, or inaccurate clinical information was provided. Inaccurate clinical data in patients with no hernia on physical exam led to a significant difference in the radiologic hernia detection rate (54.3% versus 35.7%, p = 0.007). No clinical data in patients with a hernia on physical exam led to a lower radiologic hernia detection rate (75.0% versus 93.8%, p = 0.001). CONCLUSIONS: The presence and accuracy of clinical information provided to radiologists impacts the diagnosis of abdominal wall hernias in up to 25% of cases. Standardization of both clinical and radiologic examinations for hernias and their reporting are needed. TRIAL REGISTRATION: Clinicaltrials.gov, Number NCT03121131, https://clinicaltrials.gov/ct2/show/NCT03121131.

TGF-beta repression of Id2 induces apoptosis in gut epithelial cells.

Transforming growth factor-beta (TGF-beta) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of the TGF-beta signaling pathway often occurs in colorectal cancers. Earlier, we have shown that TGF-beta induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-beta/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-beta repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE-1 cells, whereas overexpression of Id2 attenuated TGF-beta-induced apoptosis. TranSignal Protein/DNA arrays were used to identify the hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-beta. HIF-1 is a basic helix-loop-helix protein, and overexpression of Id2 blocked HIF-1 activation by TGF-beta. Furthermore, knockdown of HIF-1 blocked TGF-beta-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-beta-induced apoptosis.

Ultimate-state scaling in a shell model for homogeneous turbulent convection.

An interesting question in turbulent convection is how the heat transport depends on the strength of thermal forcing in the limit of very large thermal forcing. Kraichnan predicted [Phys. Fluids 5, 1374 (1962)] that for fluids with low Prandtl number (Pr), the heat transport measured by the Nusselt number (Nu) would depend on the strength of thermal forcing measured by the Rayleigh number (Ra) as Nu approximately Ra(1/2) with logarithmic corrections at very high Ra. According to Kraichnan, the shear boundary layers play a crucial role in giving rise to this so-called ultimate-state scaling. A similar scaling result is predicted by the Grossmann-Lohse theory [J. Fluid Mech. 407, 27 (2000)], but with the assumption that the ultimate state is a bulk-dominated state in which both the average kinetic and thermal dissipation rates are dominated by contributions from the bulk of the flow with the boundary layers either broken down or playing no role in the heat transport. In this paper, we study the dependence of Nu and the Reynolds number (Re) measuring the root-mean-squared velocity fluctuations on Ra and Pr, for low Pr, using a shell model for homogeneous turbulent convection where buoyancy is acting directly on most of the scales. We find that Nu approximately Ra(1/2)Pr(1/2) and Re approximately Ra(1/2)Pr(-1/2) , which resemble the ultimate-state scaling behavior for fluids with low Pr, and show that the presence of a drag acting on the large scales is crucial in giving rise to such scaling. As a large-scale drag cannot exist by itself in the bulk of turbulent thermal convection, our results indicate that if buoyancy acts on most of the scales in the bulk of turbulent convection at very high Ra, then the ultimate state cannot be bulk dominated.

Evi1 is a survival factor which conveys resistance to both TGFbeta- and taxol-mediated cell death via PI3K/AKT.

In hematopoietic cells the transforming potential of the ecotropic viral integration site 1 (Evi1) oncogene is thought to be dependent upon the ability to inhibit TGFbeta signaling. Although Evi1 has recently been implicated in certain epithelial cancers, the effects of Evi1 on transformation and TGFbeta signaling in epithelial cells are not completely understood. Herein, we have determined the effects of Evi1 on TGFbeta signaling in intestinal epithelial cells. Stable expression of Evi1 in non-transformed intestinal epithelial cells inhibited induction of some Smad3-dependent TGFbeta target genes, such as PAI1. However, TGFbeta-mediated induction of cellular adhesion signaling components such as integrin1 and paxillin was not inhibited by Evi1; nor did Evi1 inhibit TGFbeta-mediated epithelial to mesenchymal transition. Likewise, Evi1 did not inhibit TGFbeta-mediated downregulation of cyclin D1 or block TGFbeta-mediated growth inhibition. However, Evi1 did inhibit TGFbeta-mediated apoptosis by a process that involves phosphoinositide-3-kinase (PI3K) and its downstream effector AKT. The ability of Evi1 to suppress apoptosis is not restricted to TGFbeta-mediated cell death, since Evi1 also protects intestinal epithelial cells from taxol-mediated apoptosis. Evi1 is overexpressed in some human colon cancer cell lines, and overexpression is associated with amplification of the Evi1 gene. Knockdown of Evi1 by siRNA inhibited AKT phosphorylation in HT-29 human colon cancer cells and increased their sensitivity to taxol-mediated apoptosis. These data indicate that Evi1 functions as a survival gene in intestinal epithelial cells and colon cancer cells, activating PI3K/AKT and conveying resistance to both physiological and therapeutic apoptotic stimuli.

The role of caspases in methotrexate-induced gastrointestinal toxicity.

BACKGROUND: Enterocolitis is the major toxicity of methotrexate-based cancer chemotherapy, which limits its clinical applications. Methotrexate induces gut mucosal apoptosis in vivo; however, little is known about the molecular mechanism involved. The effectors of apoptosis include the caspase family of proteases, which are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were (1) to establish an in vitro model of methotrexate-induced gut apoptosis and (2) to determine the role of caspases in methotrexate-induced apoptosis in intestinal epithelial cells. METHODS: Rat intestinal epithelial cells (RIE-1) were treated with methotrexate in the absence or presence of ZVAD-fluoromethyl ketone, a general caspase inhibitor. Apoptosis was quantified by means of deoxyribonucleic acid (DNA) fragmentation assays and Hoechst nuclear staining. Caspase activation was measured with the use of fluorogenic substrates. RESULTS: Methotrexate induced apoptosis and decreased cell number in RIE-1 cells. DNA fragmentation was preceded by the sequential activation of caspases 9, 2, and 3, whereas caspases 1 and 8 remained inactive. ZVAD-fluoromethyl ketone inhibited methotrexate-induced caspase activation, DNA fragmentation, and nuclear condensation. CONCLUSIONS: These results indicate that methotrexate activates specific caspases and induces apoptosis in RIE-1 cells. Furthermore, caspases may play an important role in methotrexate-induced apoptosis in RIE-1 cells and may be potential therapeutic targets to attenuate methotrexate-induced enterocolitis.

Synthesis of 4-alkoxy-2-phenylquinoline derivatives as potent antiplatelet agents.

In our continuing search for novel antiplatelet agents, 4-alkoxy derivatives of 2-phenylquinoline as well as related compounds were prepared. Through biological screening, a preliminary structure antiplatelet activity relationship was established. Compounds 5-ethyl-4-methoxy-2-phenylquinoline (8), 4-ethoxy-5-ethyl-2-phenylquinoline (9), 4-ethoxycarbonylmethoxy-5-ethyl-2-phenylquinoline (10), 4-ethoxycarbonylbutoxy-5-ethyl-2-phenylquinoline (12) and 5-ethyl-4-(N-ethylcarboxido)methoxy-2-phenylquinoline (17) all demonstrated potent antiplatelet activity. Among them, compound 8 was the most potent with an IC50 value of 0.08 microM and was about 3-fold more active than indomethacin. The mechanism of antiplatelet action of 8 is possibly through its inhibition on cyclooxygenase or thromboxane synthetase.

Prevention of mucosal atrophy: role of glutamine and caspases in apoptosis in intestinal epithelial cells.

Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-1) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in RIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut.

Increased small bowel epithelial turnover in interleukin-1 receptor knockout mice.

OBJECTIVE: To determine whether interleukin-1 (IL-1) affects the cellular homeostasis of small bowel mucosa, the authors studied apoptosis and proliferation in small bowel epithelium in two groups of C57 mice: an IL-1 receptor knockout group, and a control wild-type group. SUMMARY BACKGROUND DATA: Gut mucosal integrity is maintained by a balance of cell proliferation and cell death. Recent reports suggest that IL-1, a proinflammatory cytokine, increases cell death by apoptosis in some epithelial cells. METHODS: Twenty-four male C57BL6 IL-1 receptor (type I) knockout mice were killed, and small bowel was removed for study. Twenty-four wild-type mice (C57-BL6) served as controls. Body weights, bowel length, and mucosal morphology were examined for phenotypic differences. Apoptosis was quantified by terminal deoxyuridine nick-end labeling (TUNEL) immunohistochemical staining and cellular proliferation by proliferation cell nuclear antigen staining. Whole mucosal protein was analyzed for nuclear factor-kappaB expression. Groups were analyzed by t test. RESULTS: The absence of IL-1 type I receptor in knockout mice was verified by reverse transcriptase-polymerase chain reaction. IL-1 receptor null mice were larger than wild-type controls, with a longer small bowel; however, the index of small bowel length to total body weight did not differ between groups. The percentage of apoptotic cells was higher in IL-1 receptor null mice than in wild-type mice; the proliferation index also increased. Mucosal height and other measures of mucosal morphology were not different. Genotypic absence of IL-1 receptors was associated with decreased expression of nuclear factor-kappaB in whole mucosal protein extracts. CONCLUSIONS: Both apoptosis and proliferation increased in gut epithelial cells of mice without IL-1 receptors, suggesting increased cell turnover with no change in net balance. This model represents an opportunity to examine potential mechanisms of gut epithelial turnover in vivo, under both normal conditions and in conditions of mucosal proliferation and atrophy.

A novel cytotoxic agent for human carcinoid tumors.

BACKGROUND: Conventional adjuvant therapy for advanced carcinoid tumors remains disappointing; novel therapeutic agents are needed. We have shown previously that inhibiting polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) slows the growth of carcinoid tumors. However, the clinical utility of DFMO has been limited by its cytostatic property. Synthetic polyamine analogs such as 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) appear to be cytotoxic against several human tumors. The purpose of our study was to determine whether BE-4-4-4-4 is a more effective antiproliferative and cytotoxic agent than DFMO on human carcinoid (BON) cells in vitro. METHODS: BON cells were treated with either 5 mmol/L DFMO, 0.5 to 10 mumol/L BE-4-4-4-4, or vehicle (control). Ornithine decarboxylase activity was determined by the rate of 14CO2 production, and intracellular polyamine levels were determined by chromatography. Cell number and viability were determined by Coulter counter and trypan blue exclusion, respectively. RESULTS: BE-4-4-4-4 inhibited ornithine decarboxylase activity and depleted all 3 polyamines. BE-4-4-4-4 decreased cell numbers by 81% compared with control and 27% compared with DFMO. BE-4-4-4-4 also induced a 2-fold increase in cell death compared with control or DFMO. CONCLUSIONS: BE-4-4-4-4 is cytotoxic and more effective than DFMO in inhibiting growth of BON cells. Polyamine analogs such as BE-4-4-4-4 may be effective adjuvant therapeutic agents for advanced carcinoid tumors.

A novel strategy for inhibiting growth of human pancreatic cancer cells by blocking cyclin-dependent kinase activity.

Pancreatic cancers frequently carry mutations in the K-ras, p53, and p16 genes, which regulate cell proliferation. Transition from G1 to S phase of the cell cycle requires activation of cyclin-dependent kinase 2 (Cdk2) which is inhibited by olomoucine and roscovitine. The purpose of this study was to determine whether olomoucine and roscovitine can block Cdk2 kinase activity and inhibit proliferation of four human pancreatic cancer cell lines with various genetic alterations. Human pancreatic carcinoma cell lines BxPC-3, PANC-1 Capan-2, and CAV were treated with olomoucine or roscovitine. Cdk2 kinase activity was determined using histone H1 as the substrate. Cell cycle distribution was analyzed by DNA flow cytometry. Cell numbers were quantitated by Coulter counter. Olomoucine and roscovitine blocked Cdk2 activity in all four pancreatic cancer cell lines. Both compounds also inhibited cell proliferation in a dose-dependent fashion. Roscovitine was at least threefold more potent than olomoucine for both Cdk2 activity and cell proliferation. We have shown that Cdk inhibitors, olomoucine and roscovitine, block proliferation of human pancreatic cancer cells regardless of their mutations in K-ras p53, or p16 genes. These compounds represent a novel therapeutic strategy with potential therapeutic benefits for pancreatic cancers.

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4.

The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1) in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G1/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G1/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3 postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21(Waf1/Cip1) to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21(Waf1/Cip1) in CDK2 inactivation; however, immunodepletion of p21(Waf1/Cip1) from Caco-2 protein extracts demonstrated that p21(Waf1/Cip1) is only partially responsible for CDK2 suppression at day 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G1 arrest and inhibition of proliferation that precede Caco-2 cell differentiation.

Glutamine deprivation induces apoptosis in intestinal epithelial cells.

BACKGROUND: Glutamine is the most abundant amino acid in the blood, and its deprivation leads to gut mucosal atrophy. The small intestinal mucosa is maintained by a balance between cell proliferation and cell death by apoptosis. We reported that glutamine is required for nitrogen-stimulated proliferation in intestinal epithelial cells. We do not know whether glutamine regulates apoptosis in the gut. The purpose of this study is to determine whether glutamine deprivation induces apoptosis in rat intestinal epithelial (RIE-1) cells and to compare the effect of glutamine starvation with that of methionine and cysteine (Met/Cys) starvation. METHODS: RIE-1 cells were deprived of either glutamine or Met/Cys for 24 hours. Cell numbers were determined by cell counting and tetrazolium enzymatic assay. Apoptosis was quantified by Annexin V assay and confirmed by DNA gel electrophoresis and Hoecsht nuclear staining. RESULTS: Deprivation of glutamine or Met/Cys resulted in decreased cell numbers. However, only the glutamine-deprived group showed significant induction of apoptosis with increased Annexin V staining, DNA laddering, and nuclear condensation. CONCLUSIONS: This study provides biochemical and morphologic evidence that glutamine deprivation induces apoptosis in rat intestinal epithelial cells. In contrast, Met/Cys starvation suppresses cell number without induction of apoptosis. These results suggest that glutamine serves as a specific survival factor in enterocytes.

Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1.

BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression.

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.

Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase.

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.

Effectiveness of non-mydriatic retinal photography and direct ophthalmoscopy in detecting diabetic retinopathy.

This is a prospective study to compare the effectiveness of non-mydriatic photography with that of direct ophthalmoscopy in screening for diabetic retinopathy in 153 patients attending a hospital clinic in Hong Kong. Retinal photography under physiological mydriasis and direct ophthalmoscopy of patients with dilated pupils were compared with the ophthalmologists' examination results as a reference standard. The prevalence of diabetic retinopathy in this sample population was 15%. The sensitivity of detecting diabetic retinopathy by retinal photography was higher than that of direct ophthalmoscopy (64% versus 41%; 95% confidence interval of difference, 1.2%- 44.3%). Of five patients who had serious retinopathy, retinal photography failed to detect the disease in two; direct ophthalmoscopy failed to detect the disease in all five patients. Specificities of retinal photography and direct ophthalmoscopy were 90% (95% confidence interval, 84%-96%) and 93% (95% confidence interval, 88%-97%), respectively. We conclude that retinal photography is significantly more effective than direct ophthalmoscopy in detecting diabetic retinopathy. In addition, the non-mydriatic camera is easy to use and is the preferred method of screening.

Cell cycle-mediated regulation of hepatic regeneration.

BACKGROUND: Hepatic regeneration after partial hepatectomy (PH) is characterized by a synchronous induction of normally quiescent hepatocytes to reenter the cell cycle, leading to a complete restoration of hepatic mass. Cell cycle progression requires activation of cyclin-dependent kinases (Cdks) that are regulated by cyclins and Cdk inhibitors. METHODS: Protein expression of the cyclins (D-type and E), Cdks (Cdk2 and 4), and Cdk inhibitors (p21 and p27) was measured by Western blot after SHAM operation or PH in F344 rats. In addition, Cdk2-associated kinase activity was measured. RESULTS: Rapid induction of D-type and E cyclins, as well as their catalytic partners, Cdk2 and Cdk4, occurred after PH in rats. Complexes containing cyclin E and Cdk2 assembled in the regenerating liver, leading to increased Cdk2-associated kinase activity. The regenerating liver returned to preresection weight by day 7, at which time the Cdk2 activity also returned to SHAM levels. Biphasic induction of the Cdk inhibitor p21 was observed; the first peak occurred as early as 6 hours after PH, with a subsequent peak in expression occurring at 24 to 72 hours after PH. CONCLUSIONS: Taken together, these data support the concept that cyclins, Cdks, and Cdk inhibitors regulate cell cycle progression in the regenerating liver. In addition, the induction of p21 at two time points suggests that this protein may regulate both early proliferation and subsequent inhibition of hepatocyte regeneration.

Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells.

Examination of a panel of ER positive breast cancer cell lines showed that they were differentially growth inhibited by vitamin D3 and its analogue EB1089. EB1089 treatment of the breast cancer cell lines MCF-7 E, BT20, T47D, and ZR75 demonstrated a correlation between a reduction in Cdk2 kinase activity towards phosphorylation of histone H1 and a decrease in DNA synthesis, while no modulation of Cdk2 activity was observed in the vitamin D3 and EB1089 resistant cell line MCF-7 L. This was accompanied by a time dependent decrease in the percentage of S phase cells in the responsive lines. Characterization of the expression levels of Cdk2 and its related cell cycle proteins in MCF-7 E cells showed that after EB1089 treatment, there was a concentration and time dependent up-regulation of p21 as well as a decrease in cyclin A proteins. Paradoxically, cyclin E levels were increased as a function of treatment. Analysis of cyclin-Cdk2-Cdki complex formation showed that in EB1089 treated MCF-7 E cells, Cdk2, cyclin A and cyclin E immunoprecipitates contained an increased abundance of p21. In contrast to MCF-7 E cells, increases in both p21 and p27 as well as their complex formation with Cdk2 were observed in BT20 and ZR75 cells. These findings indicate that up-regulation of p21 as well as p27 in some cell types may account for the inactivation of Cdk2 activity and a G1 block of the cell cycle following EB1089 treatment.

Cyclin-dependent kinase inhibitors block proliferation of human gastric cancer cells.

BACKGROUND: Olomoucine and roscovitine are novel compounds that are designed to inhibit cyclin-dependent kinases (e.g., Cdk2 and cdc2). Cdks regulate progression through key checkpoints of the cell cycle. The purpose of this study was to determine (1) whether olomoucine and roscovitine inhibit Cdk2 and cdc2 kinase activities of the human gastric cancer cell line SIIA and (2) whether olomoucine and roscovitine block cell proliferation and cell cycle progression. METHODS: SIIA cells were treated with olomoucine or roscovitine and examined for Cdk2 and cdc2 activities by using histone H1 as the substrate. Cell numbers were counted with a Coulter counter. Cell cycle distribution was analyzed by DNA flow cytometry. RESULTS: Olomoucine and roscovitine completely blocked Cdk2 and cdc2 activities in SIIA cells. Both compounds were also able to inhibit proliferation of SIIA cells, as well as three other human gastric cancer cell lines (AGS, MKN45-630, and SNU-1). Cell cycle analysis showed that treatment with olomoucine or roscovitine for 24 hours led to a decrease in the S phase population and an increase in the G2/M population. CONCLUSIONS: We have shown that Cdk inhibitors, olomoucine and roscovitine, are a new class of antineoplastic molecules with potential therapeutic benefits for gastric cancers.