Comparison of joint degeneration and pain in male and female mice in DMM model of osteoarthritis.

OBJECTIVE: While the prevalence of radiographic and symptomatic osteoarthritis (OA) is higher in women, male mice are more frequently used in animal experiments to explore its pathogenesis or drug efficacy. In this study, we examined whether sexual dimorphism affects pain and joint degeneration in destabilization of the medial meniscus (DMM) mouse model. METHODS: DMM or sham surgery was performed on the knee of male and female C57BL/6 mice. Joint damage was assessed by safranin O staining and scored using the Osteoarthritis Research Society International (OARSI) scoring system. Von Frey hair, incapacitance, and rotarod tests were conducted to measure joint pain. The analgesic effect of capsazepine (CPZ), a TRPV1 antagonist, was compared between male and female mice. RESULTS: Histology and OARSI scoring analysis showed that cartilage degeneration developed, and progressed in both male and female DMM groups, however, damage was less severe in females at the late stage of OA. Pain behavior, as measured by mechanical allodynia, was displayed for longer in male DMM mice compared to females. Incapacitance data showed that CPZ significantly reduced DMM-induced pain in male mice but not in female mice. Immunofluorescence microscopy analysis demonstrated that DMM surgery increased the expression of TRPV1 in both female and male dorsal root ganglion (DRG). Injection of CPZ significantly suppressed TRPV1 expression in the DRG of male mice only. CONCLUSION: Joint damage develops comparably in both female and male mice after DMM although it progresses less in females. There was a subtle sex difference in pain behaviors and analgesic efficacy of a TRPV1 antagonist, which was accompanied by a differential regulation of TPRV1.

Vaginal compared with intramuscular progestogen for preventing preterm birth in high-risk pregnant women (VICTORIA study): a multicentre, open-label randomised trial and meta-analysis.

OBJECTIVE: To compare the efficacy of two types of progestogen therapy for preventing preterm birth (PTB) and to review the relevant literature. DESIGN: A multicentre, randomised, open-label, equivalence trial and a meta-analysis. SETTING: Tertiary referral hospitals in South Korea. POPULATION: Pregnant women with a history of spontaneous PTB or short cervical length (<25 mm). METHODS: Eligible women were screened and randomised at 16-22 weeks of gestation to receive either 200 mg of vaginal micronised progesterone daily (vaginal group) or an intramuscular injection of 250 mg 17α-hydroxyprogesterone caproate weekly (IM group). Stratified randomisation was carried out according to participating centres and indications for progestogen therapy. This trial was registered at ClinicalTrials.gov (NCT02304237). MAIN OUTCOME MEASURE: Preterm birth (PTB) before 37 weeks of gestation. RESULTS: A total of 266 women were randomly assigned and a total of 247 women (119 and 128 women in the vaginal and IM groups, respectively) were available for the intention-to-treat analysis. Risks of PTB before 37 weeks of gestation did not significantly differ between the two groups (22.7 versus 25.8%, P = 0.571). The difference in PTB risk between the two groups was 3.1% (95% CI -7.6 to 13.8%), which was within the equivalence margin of 15%. The meta-analysis results showed no significant differences in the risk of PTB between the vaginal and IM progestogen treatments. CONCLUSION: Compared with vaginal progesterone, treatment with intramuscular progestin might increase the risk of PTB before 37 weeks of gestation by as much as 13.8%, or reduce the risk by as much as 7.6%, in women with a history of spontaneous PTB or with short cervical length. TWEETABLE ABSTRACT: Vaginal and intramuscular progestogen showed equivalent efficacy for preventing preterm birth before 37 weeks of gestation.

Fibronectin fragment inhibits xylosyltransferase-1 expression by regulating Sp1/Sp3- dependent transcription in articular chondrocytes.

OBJECTIVE: We investigated the effects of 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) on xylosyltransferase-1 (XT-1), an essential anabolic enzyme that catalyzes the initial and rate-determining step in glycosaminoglycan chain synthesis, in human primary chondrocytes. METHODS: Proteoglycan and XT-1 expression in cartilage tissue was analyzed using safranin O staining and immunohistochemistry. The effects of 29-kDa FN-f on XT-1 expression and its relevant signaling pathway were analyzed by quantitative real-time-PCR, immunoblotting, chromatin immunoprecipitation, and immunoprecipitation assays. The receptors for 29-kDa FN-f were investigated using small interference RNA and blocking antibodies. RESULTS: The expression of XT-1 was significantly lower in human osteoarthritis cartilage than in normal cartilage. Intra-articular injection of 29-kDa FN-f reduced proteoglycan levels and XT-1 expression in murine cartilage. In addition, in 29-kDa FN-f-treated cells, XT-1 expression was significantly suppressed at both the mRNA and protein levels, modulated by the transcription factors specificity protein 1 (Sp1), Sp3, and activator protein 1 (AP-1). The 29-kDa FN-f suppressed the binding of Sp1 to the promoter region of XT-1 and enhanced the binding of Sp3 and AP-1. Inhibition of mitogen-activated protein kinase and nuclear factor kappa B signaling pathways restored the 29-kDa FN-f-inhibited XT-1 expression, along with the altered expression of Sp1 and Sp3. Blockading toll-like receptor 2 (TLR-2) and integrin α5ß1 via siRNA and blocking antibodies revealed that the effects of 29-kDa FN-f on XT-1 expression were mediated through the TLR-2 and integrin α5ß1 signaling pathways. CONCLUSION: These results demonstrate that 29-kDa FN-f negatively affects cartilage anabolism by regulating glycosaminoglycan formation through XT-1.

TLR5 activation induces expression of the pro-inflammatory mediator Urokinase Plasminogen Activator via NF-κB and MAPK signalling pathways in human dental pulp cells.

AIM: To explore the involvement of TLR5 in pulp inflammation and to examine the effects of TLR5 activation with its ligand, FlaB protein, on pro-inflammatory gene expression. METHODOLOGY: TLR5 expression in dental pulp tissues and human dental pulp cells (hDPCs) were determined by immunohistochemistry, immunocytochemistry, Western blots and RT-PCR analyses. To examine the role of TLR5, hDPCs were treated with recombinant FlaB protein (500 ng mL-1 ) to activate the receptor or with a small interfering RNA against TLR5 (si-TLR5) to downregulate the receptor. After exposure to FlaB, the expression of inflammation-related proteins was screened using a protein array kit. Western blots or qRT-PCR analyses were performed to identify changes in the expression of uPA (urokinase plasminogen activator), TIMPs (tissue inhibitor of metalloproteinases), and IL-6 and to determine their signalling pathways. Statistical analysis was performed using one-way analysis of variance (anova) with Tukey post hoc test; P < 0.05 was considered statistically significant. RESULT: TLR5 expression was identified in pulp tissues and hDPCs. In the protein array analysis, treatment with FlaB significantly increased uPA expression (P < 0.01) and significantly decreased TIMP1/4 (P < 0.05). FlaB treatment also significantly increased expression of the inflammatory marker IL-6 (P < 0.01). FlaB treatment increased phosphorylation of the NF-κB p65 subunit, JNK, p38 and ERK. Chemical inhibitors of NF-κB (Bay11-7082), p38 (SB202190) or ERK (U0126) decreased the FlaB induction of uPA expression. Downregulation of TLR5 expression by siRNA decreased the FlaB induction of uPA protein and p65 phosphorylation. CONCLUSION: TLR5 activation with FlaB treatment induced the expression of uPA via the NF-κB and MAPK signalling pathways. Flagellin-bearing oral bacteria may cause pulp inflammation through TLR5. The findings provide new clues to control pulpal diseases by targeting TLR5 signalling pathways.

Clinical impact of coexisting retinopathy and vascular calcification on chronic kidney disease progression and cardiovascular events.

BACKGROUND AND AIMS: Retinopathy and vascular calcification (VC) are representative markers of microvascular and macrovascular dysfunction in patients with chronic kidney disease (CKD). However, their relationship and combined effects on clinical outcomes remain undetermined. METHODS AND RESULTS: We included 523 patients with nondialysis-dependent CKD stage 3-5 who had been examined with fundus photography for diabetic or hypertensive retinopathy. Simple radiographs were analyzed for the presence of VC. The clinical significance of VC of the abdominal aorta and iliofemoral artery (apVC) and retinopathy was evaluated in terms of the rate of renal function decline and composite of any cardiovascular event or death. CKD patients with retinopathy showed higher prevalence of apVC than those without retinopathy (25.6% vs. 12.5%, P < 0.001).The presence of retinopathy was independently associated with apVC (OR 2.13, 95% CI 1.31, 3.49). In multivariate analysis, compared with subjects with neither apVC nor retinopathy, the coexistence of both apVC and retinopathy were independently associated with rapid renal function decline (ß = -1.51; 95% CI -2.40, -0.61), whereas apVC or retinopathy alone were not. Compared with subjects with neither apVC nor retinopathy, the HRs for composite end points were 1.05 (95% CI 0.48, 2.27), 1.79 (95% CI 1.14, 2.80), and 2.07 (95% CI 1.17, 3.67) for patients with apVC only, those with retinopathy only, and those with both apVC and retinopathy, respectively. CONCLUSION: The coexistence of VC and retinopathy was independently associated with CKD progression and cardiovascular events or deaths, and its combined effect was stronger than any separate condition.

Differential identification of three species of Curtovirus using loop-mediated isothermal amplification.

Rapid and sensitive detection methods for three species of Curtovirus were developed using a loop-mediated isothermal amplification (LAMP) technique. A universal primer set for detecting the three main species of Curtovirus at the same time, and three kinds of species-specific primer sets were designed and used for LAMP reactions. Results from the LAMP reactions were visualized both by color changes after adding SYBR Green I staining dye and by DNA laddering on agarose gel electrophoresis. The optimal conditions for the curtovirus LAMP reaction were confirmed at 60°C for the universal primers and at 62°C for the three species-specific primer sets. Amplification of curtoviruses by LAMP reaction was ten-fold more sensitive than that by polymerase chain reaction. Primers designed for curtovirus detection in this study did not anneal to or amplify DNA from other DNA or RNA viruses (tomato yellow leaf curl virus, tomato spotted wilt virus, and potato virus Y). Taken together, the primer sets and reaction conditions developed in this study show that the LAMP technique could be a useful tool to detect the three species of Curtovirus simultaneously and distinguish them in the laboratory and the field.

Application of orthodontic forces prior to autotransplantation - case reports.

AIM: This case report describes the successful autotransplantation of mandibular molars after application of orthodontic forces and discusses the advantages of this technique, that is, pre-application of an orthodontic force for autotransplantation. SUMMARY: After clinical and radiographic examination, autotransplantation was planned with the patient's written informed consent. An orthodontic force was applied, and the surgical procedure was performed after tooth mobility had increased. Root canal treatment was performed within 2 weeks of autotransplantation. At the 1-year follow-up, the transplanted teeth revealed asymptomatic and healthy periodontal conditions. KEY LEARNING POINTS: Autotransplantation is the surgical movement of a tooth from its original location to another site. The pre-application of orthodontic force technique was recently introduced for autogenous tooth transplantation. Pre-application of an orthodontic force may be a useful treatment option for autotransplantation.

A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease-like 2.

We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.

Maternal serum highly sensitive C-reactive protein in normal pregnancy and pre-eclampsia.

OBJECTIVE: To establish reference values for highly sensitive C-reactive protein (hsCRP) in normal pregnancy and determine whether disease severity could be predicted by serum hsCRP concentration in women with severe pre-eclampsia (PE). METHODS: Blood samples were collected from 25 pregnant women with severe PE and 202 healthy pregnant women who were divided into 4 groups by pregnancy duration. Levels of hsCRP were determined by the immunonephelometric method. Mean arterial pressure, time between delivery and diuresis, and mean ratio of systolic to diastolic blood flow velocity in uterine arteries were used as indicators of the severity of PE. RESULTS: The median values of hsCRP in each group were 0.76 mg/L (0.16-13.61 mg/L), 1.53 mg/L (0.39-20.31 mg/L), 2.08 mg/L (0.50-9.45 mg/L), and 2.28 mg/L (0.44-8.11 mg/L) and showed a trend toward increase. Serum levels of hsCRP were positively correlated with each severity indicator of PE. CONCLUSIONS: This study showed that hsCRP levels were positively correlated to pregnancy duration in healthy women and could be used as a severity marker in women with severe PE.

Lacrimal gland-derived IL-22 regulates IL-17-mediated ocular mucosal inflammation.

Inflammatory damage of mucosal surface of the eye is a hallmark of dry eye disease (DED) and, in severe cases, can lead to significant discomfort, visual impairment, and blindness. DED is a multifactorial autoimmune disorder with a largely unknown pathogenesis. Using a cross-sectional patient study and a well-characterized murine model of DED, herein we investigated the immunoregulatory function of interleukin-22 (IL-22) in the pathogenesis of DED. We found that IL-22 levels were elevated in lacrimal fluids of DED patients and inversely correlated with severity of disease. Acinar cells of the lacrimal glands (LGs), not inflammatory immune cells, are the primary source of IL-22, which suppresses inflammation in ocular surface epithelial cells upon desiccating stress. Moreover, loss of function analyses using IL-22 knockout mice demonstrated that IL-22 is essential for suppression of ocular surface infiltration of Th17 cells and inhibition of DED induction. Our novel findings elucidate immunoregulatory function of LG-derived IL-22 in inhibiting IL-17-mediated ocular surface epitheliopathy in DED thus making IL-22 a new relevant therapeutic target.

Water quality modeling to evaluate BMPs in rice paddies.

A water quality model applicable to rice paddies was developed using field data from 1999-2002. Use of the Dirac delta function efficiently explained the nutrient-concentration characteristics of ponded water. The model results agreed reasonably well with the observed data. The ponded-water quality was influenced primarily by fertilization; nutrient concentration was especially high during early cultivation periods. Reducing surface drainage during the fertilization period may substantially reduce nonpoint source loading from paddies. Increased weir heights and shallow irrigation methods were evaluated by the model as practical methods for reducing nutrient loading from paddies. These methods were effective in reducing surface drainage and are suggested as "best management practices" (BMPs) if applied based on site-specific paddy conditions.

Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization.

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.

Role of the hMLH1 DNA mismatch repair protein in fluoropyrimidine-mediated cell death and cell cycle responses.

DNA mismatch repair (MMR) is an efficient system for the detection and repair of mismatched and unpaired bases in DNA. Deficiencies in MMR are commonly found in both hereditary and sporadic colorectal cancers, as well as in cancers of other tissues. Because fluorinated thymidine analogues (which through their actions might generate lesions recognizable by MMR) are widely used in the treatment of colorectal cancer, we investigated the role of MMR in cellular responses to 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd). Human MLH1(-) and MMR-deficient HCT116 colon cancer cells were 18-fold more resistant to 7.5 microM 5-fluorouracil (continuous treatment) and 17-fold more resistant to 7.5 microM FdUrd in clonogenic survival assays compared with genetically matched, MLH1(+) and MMR-proficient HCT116 3-6 cells. Likewise, murine MLH1(-) and MMR-deficient CT-5 cells were 3-fold more resistant to a 2-h pulse of 10 microM FdUrd than their MLH1(+) and MMR-proficient ME-10 counterparts. Decreased cytotoxicity in MMR-deficient cells after treatment with various methylating agents and other base analogues has been well reported and is believed to reflect a tolerance to DNA damage. Synchronized HCT116 3-6 cells treated with a low dose of FdUrd had a 2-fold greater G(2) cell cycle arrest compared with MMR-deficient HCT116 cells, and asynchronous ME-10 cells demonstrated a 4-fold greater G(2) arrest after FdUrd treatment compared with CT-5 cells. Enhanced G(2) arrest in MMR-proficient cells in response to other agents has been reported and is believed to allow time for DNA repair. G(2) cell cycle arrest as determined by propidium iodide staining was not a result of mitotic arrest, but rather a true G(2) arrest, as indicated by elevated cyclin B1 levels and a lack of staining with mitotic protein monoclonal antibody 2. Additionally, p53 and GADD45 levels were induced in FdUrd-treated HCT116 3-6 cells. DNA double-strand break (DSB) formation was 2-fold higher in MMR-proficient HCT116 3-6 cells after FdUrd treatment, as determined by pulsed-field gel electrophoresis. The formation of DSBs was not the result of enhanced apoptosis in MMR-proficient cells. FdUrd-mediated cytotoxicity was caused by DNA-directed and not RNA-directed effects, because administration of excess thymidine (and not uridine) prevented cytotoxicity, cell cycle arrest, and DSB formation. hMLH1-dependent responses to fluoropyrimidine treatment, which may involve the action of p53 and the formation of DSBs, clearly have clinical relevance for the use of this class of drugs in the treatment of tumors with MMR deficiencies.

The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells.

Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1+) and MMR-deficient (hMLH1-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1+) cells compared to HCT116 (hMLH1-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1+/+ spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity.

Selective radiosensitization of drug-resistant MutS homologue-2 (MSH2) mismatch repair-deficient cells by halogenated thymidine (dThd) analogues: Msh2 mediates dThd analogue DNA levels and the differential cytotoxicity and cell cycle effects of the dThd analogues and 6-thioguanine.

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2+/+ and Msh2-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2+/+ and Msh2-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2+/+ and human HEC59/2-4 (MSH2+) MMR-proficient cells, whereas Msh2-/- cells and human HEC59 (MSH2-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2+/+ ES-derived and HEC59/2-4 cells to IdUrd, whereas Msh2-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2+ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2+/+ and Msh2-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G2-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.

Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae.

The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity. We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation. IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe. IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency [1]. However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation. In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei. In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells. The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds. Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth. Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more. A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB). The cross-species expressions of IBD1 in S. pombe and byr4 in S. cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes. We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.

Dysfunction of insulin mediator protein kinase FA in lymphocytes of patients with NIDDM.

Patients with non-insulin-dependent diabetes mellitus (NIDDM) had an impaired capability to activate exogenous ATP.Mg-dependent protein phosphatase in lymphocytes compared with nondiabetic subjects. More importantly, the impaired protein phosphatase activation in the lymphocytes of patients with NIDDM could be consistently and completely restored to normal by exogenous pure protein kinase FA (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in patients with NIDDM is due to a functional loss of kinase FA. By contrast, both NIDDM patients and nondiabetic subjects had similar levels of total cell proteins and spontaneously active protein phosphatase activity in their lymphocytes, indicating that the dysfunction of kinase FA in patients with NIDDM is very specific. Statistical analysis further revealed that the lymphocytes isolated from 21 nondiabetic subjects contained high levels of FA activity (148 +/- 22 mU/mg cell protein), whereas, the lymphocytes of 21 patients with NIDDM contained low levels of FA activity (50 +/- 22 mU/mg), indicating statistically significant differences in FA activity between diabetic patients and nondiabetic subjects. This is the first report providing initial evidence that patients with NIDDM may statistically have a common impairment in the protein phosphatase activation in their lymphocytes and that the molecular mechanism for this defect is due to a biochemical dysfunction of protein kinase FA, a biological mediator for both insulin and epidermal growth factor.

Preclinical study of the systemic toxicity and pharmacokinetics of 5-iodo-2-deoxypyrimidinone-2'-deoxyribose as a radiosensitizing prodrug in two, non-rodent animal species: implications for phase I study design.

We have demonstrated previously an improved therapeutic index for oral 5-iodo-2-deoxypyrimidinone-2'-deoxyribose (IPdR) compared with oral and continuous infusion of 5-iodo-2'-deoxyuridine (IUdR) as a radiosensitizing agent using three different human tumor xenografts in athymic mice. IPdR is a prodrug that is efficiently converted to IUdR by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for > or =1 h after p.o. IPdR. Athymic mice tolerated oral IPdR at up to 1500 mg/kg/day given four times per day for 6-14 days without significant systemic toxicities. In anticipation of an investigational new drug application for the first clinical Phase I and pharmacology study of oral IPdR in humans, we studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species. For the IPdR systemic toxicity and toxicology study, twenty-four male or female ferrets were randomly assigned to four IPdR dosage groups receiving 0, 15, 150, and 1500 mg/kg/day by oral gavage x 14 days prior to sacrifice on study day 15. All ferrets survived the 14-day treatment. Ferrets receiving 1500 mg/kg/day showed observable systemic toxicities with diarrhea, emesis, weight loss, and decreased motor activity beginning at days 5-8 of the 14-day schedule. Overall, both male and female ferrets receiving IPdR at 1500 mg/kg/day experienced significant weight loss (9 and 19%, respectively) compared with controls after the 14-day treatment. No weight loss or other systemic toxicities were observed in other IPdR dosage groups. Grossly, no anatomical lesions were noted at complete necropsy, although liver weights were increased in both male and female ferrets in the two higher IPdR dosage groups. Histologically, IPdR-treated animals showed dose-dependent microscopic changes in liver consisting of minimal to moderate cytoplasmic vacuolation of hepatocytes, which either occurred in the periportal area (high dosage group) or diffusely throughout the liver (lower dosage groups). Female ferrets in the highest IPdR dose group also showed decreased kidney and uterus weights at autopsy without any associated histological changes. No histological changes were found in central nervous system tissues. No significant abnormalities in blood cell counts, liver function tests, kidney function tests, or urinalysis were noted. Hepatic aldehyde oxidase activity was decreased to approximately 50 and 30% of control ferrets in the two higher IPdR dosage groups, respectively, after the 14-day treatment period. The % IUdR-DNA incorporation in ferret bone marrow at the completion of IPdR treatment was < or =0.05% in the two lower dosage groups and approximately 2% in the 1500 mg/kg/day dosage group. The % IUdR-DNA in normal liver was < or =0.05% in all IPdR dosage groups. In a pharmacokinetic study in four Rhesus monkeys, we determined the plasma concentrations of IPdR after a single i.v. bolus of 50 mg/kg over 20 min. Using a two-compartment model to fit the plasma pharmacokinetic data, we found that IPdR was cleared in these non-human primates in a biexponential manner with an initial rapid distributive phase (mean T1/2alpha = 6.5 min), followed by an elimination phase with a mean T1/2 of 63 min. The mean maximum plasma concentration of IPdR was 124+/-43 microM with a mean total body clearance of 1.75+/-0.95 l/h/kg. IPdR was below detection (<0.5 microM) in the cerebrospinal fluid. We conclude that there are dose-limiting systemic toxicities to a 14-day schedule of p.o. IPdR at 1500 mg/kg/day in ferrets that were not found previously in athymic mice. However, no significant hematological, biochemical, or histopathological changes were found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent in ferret liver, suggesting partial enzyme saturation by this IPdR schedule. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance is similar to our published data in athymic mice. These data are being applied