Comparing AMH, AFC and FSH for predicting high ovarian response in women undergoing antagonist protoco1l

JOURNAL OF MEDICAL RESEARCH MITIGATION OF IRRADIATION EFFECTS ON TASTE EPITHELIUM IN THE PROTEIN KINASE C DELTA NULL MOUSE Nguyen Manh Ha , Nguyen Xuan Hoi Hanoi Medical University, 2National Obstetrics and Gynaecology Hospital Radiotherapy for head and neck cancer typically leads to loss of taste among cancer patients We proposed that loss of taste after irradiation may be due to continued natural taste cell death, paired with temporary interruption of cell replacement One possible strategy for averting taste loss may be to reduce epithelial cell death, so that cell proliferation is not interrupted Protein kinase C delta (PKCδ) has been shown to positively regulate apoptosis and/or cell cycle arrest The aim of this study was to test whether the effects of radiation on taste epithelium are mitigated in PKCδ null mice The heads of wild type and PKCδ null adult mice were irradiated with a single 8Gy dose, and the lingual epithelia were examined for proliferative activity (Ki67 - ir) at progressive days post-irradiation (dpi) Our initial results showed that the dramatic reduction in the proliferative index typically observed in the taste epithelia of irradiated wild type mice - days after irradiation was mitigated significantly by loss of PKCδ in the PKCδ null mice Our data suggest that PKCδ may be required for apoptotic cell death and/or cell cycle arrest in irradiated taste epithelium Keywords: stem cells, transit amplifying cells, proliferation, taste loss, protein kinase C delta I INTRODUCTION Protein kinase C (PKC) belongs to a large PKCδ is activated by a variety of stimuli, family of phosphatidylserine - dependent including serine/threonine protein kinases The many drugs, oxidative stress, ultraviolet radiation, isoforms of PKC have been categorized into and organelle poisons [2] PKCδ promotes subfamilies: classical (α, βI, βII, and γ), novel apoptosis by activating many proteins in the (δ, ε, η, and θ) and atypical (λ and ζ) The apoptotic cascade, including p53, caspase 9, different isoforms of PKC play multifaceted caspase 3, caspase 7, Mcl-1 and lamin B roles in cellular response in a range of tissues Some studies have shown that PKCδ also Among the many isoforms of PKC, at least five functions (α, δ, ε, η, and ζ) are expressed in epidermal PKCδ catalytic fragment has a critical role in keratinocytes [1]; in fact, PKCδ was originally enforcing the G2/M checkpoint in response to discovered UV radiation in human keratinocytes [3] in the mouse epidermis Upon activation, PKCδ has been shown to be a key regulator of the intrinsic (mitochondrial- dependent) apoptotic pathway ionizing in cell radiation, cycle anti-cancer regulation The The loss or suppression of PKCδ has been shown to protect cells after irradiation in vivo and in vitro In the parotid gland of PKCδ null mice, apoptosis is suppressed by greater than Corresponding author: Nguyen Xuan Hoi, National Obstetrics and Gynecology Hospital E-mail: doctorhoi@gmail.com Received: 20 October 2016 Accepted: 10 December 2016 66 60% [4] Suppression of PKCδ in vitro and genetic loss in vivo also protects the salivary gland from cell death [5] ATM is involved in PKCδ regulation in response to radiation [6], JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH and ATM−/− thymic lymphoma cells in mice are more resistant to radiation - induced apoptosis than wild - type mice [7] Finally, the absence of PKCδ in PKCδ null mice reduces reperfusion injury following transient ischemia [8] Previous studies have shown that radiation targets the taste progenitor pool, composed of tbSCs and TAs, by arresting actively dividing cells and inducing apoptosis We hypothesized that the loss of PKCδ might protect taste progenitor cells from death and/or promote their continued mitosis after irradiation In this study, we firstly assessed whether PKCδ null mice possess normal taste epithelia To this, we analyzed the proliferation index and taste receptor cells of PKCδ null mice and compared these numbers with those of wild type mice Secondly, we tested whether PKCδ null mice have grossly normal taste behavior with regards to bitter tastes, using a standard two-bottle taste preference test Thirdly, we assessed whether the loss of PKCδ protects taste progenitors from death and/or cell cycle arrest by analyzing the proliferation index of taste epithelia in knockout mice at different times after irradiation, and comparing their proliferation indices to those of wild-type mice Next, we studied whether the maintenance of taste progenitor proliferation in PKCδ null mice is due to a block in cell death and/or a block in cell cycle arrest after irradiation The final aim of this study was to assess the protective effects of PKCδ on taste loss after irradiation Tissue preparation and immunostaining Mice were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer (PFA) Tongues were dissected from the lower jaw, and postfixed in 4% PFA overnight at 4°C, followed by immersion in sucrose (20% in 0.1M PB) overnight at 4°C Cryoprotected tongues were embedded in OCT compounds (Tissue Tek) and cryosectioned at 12 µm Sections were thaw-mounted and stored at -20°C overnight before staining For Ki-67 immunofluorescence, sections were washed three times in 0.1M PBS, treated with sodium citrate buffer (pH 6.0) at 95°C for 15 minutes, and cooled to room temperature for 30 minutes Sections were then washed in PBS, blocked in blocking solution with 5% normal goat serum for hours at room temperature, then soaked for 15 minutes each with avidin/biotin blocking solutions (A/B blocking kit; Vector Laboratories) Sections were then incubated in rabbit anti - Ki - 67 antiserum (1: 200; Thermo Scientific) overnight at 4°C After washing three times with PBS buffer for one hour each, sections were incubated with biotin-conjugated anti-rabbit IgG (Vector Laboratories), followed by dilution to 1:500 in PBS with 0.1% Tween 20 and 2.5% normal goat serum for one hour at room temperature The sections were washed with PBS for one hour, then incubated in Streptavidin 546 (1:1000; Chemicon International) in PBS buffer for two hours at room temperature After a final wash in PBS for one II SUBJECTS AND METHODS hour, sections were counter-stained with Sytox Animals green (Invitrogen) and mounted in Fluoro-/ Two- to four-month-old C57Bl/6 and PKCδ mount G (SouthernBiotech) - mice were used in all experiments Methods JMR 105 E1 (7) - 2016 Isolation of lingual epithelium for western blot analysis of PKCδ 67 JOURNAL OF MEDICAL RESEARCH The method that we used was adapted and mM Preference scores were defined as from Behe et al and Vandenbeuch et al [9] ratios of the total solution consumed in tube Adult mice were killed by CO2 inhalation and divided by the total solution consumed in cervical dislocation Tongues from the wild tubes and type mice were quickly removed and stored for Analysis several minutes in ice-cold Tyrode’s solution equilibrated with 100% oxygen Tyrode’s solu- To analyze the data, we obtained images, tion contains 140 nM NaCl, nM KCl, nM counted cells, and calculated the proliferation MgCl2, nM CaCl2, 10 nM HEPES, 10 nM index using Ki-67-ir The data for the two glucose, and nM Na pyruvate, pH 7.4 Lin- bottle taste preference test was gathered from gual epithelium containing taste buds were three C57Bl/6 mice and three PKCδ null mice pealed off of the tongue’s muscle, rinsed in For the immunoblot, epithelia were collected Ca2+/Mg2+-free Tyrode’s, and stored at -800C from three C57Bl/6 and PKCδ null mice Data for Immunoblot for PKCδ immunofluorescence staining were collected from three to four mice per time The method for western immunoblot for point All cell counts were corrected with the PKCδ has been described elsewhere [4] Abercrombie correction factor to account for Briefly, the circumvallate papillae and non- any variability in cell size following irradiation taste epithelium were taken from three wild treatment A T-test or way ANOVA was used type mice Protein was suspended in JNK lysis to analyze data buffer and Rabbit polyclonal antibody (Santa Cruz) was used to detect PKCδ in a 1: 1000 diluted solution III RESULTS Characterization of taste epithelium in PKCδ mice Two-bottle preference test Preferences for water versus different con- To examine whether PKCδ was expressed centrations of denatonium (which contains a in the lingual epithelium of the wild type mice, bitter taste) were tested in wild type and PKCδ we performed an immunoblot of the lingual null mice using conventional methods [10] epithelium to detect Two tubes were used, one which contained found that PKCδ was present in both the non- either different taste epithelium (NT) and the taste epithelium concentrations (tube 1) and the other which of the circumvallate papilla (CV; Figure 1A) contained water (tube 2) The mice were given Next, to assess whether the proliferative activ- 48-hour access to two drinking tubes The ity of taste epithelium in PKCδ null mice was positions of the tubes were randomized and comparable to that of wild type mice, we switched after 24 hours to avoid place prefe- employed Ki-67-IR, which marks actively rences Consumption of denatonium solution proliferating cells in all phases of the cell cycle and water was recorded for the entire 48-hour except for early G1 and G0 In PKCδ null period The following concentrations of dena- mice, actively dividing cells were detected in tonium were used: 0.03 mM; 0.1 mM; mM; the basal epithelium and in perigemmal re- 68 water or denatonium at the PKCδ protein We JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH gions around taste profiles (Figure 1C), similar wild type mice and 0.81 +/- 0.02 for the PKCδ to the pattern in wild type mice (Figure 1B) null mice These indices were not significantly The proliferation indices of Ki-67 for basal different (Figure 1D: mean +/- SD, n = - taste epithelial cells were 0.79 +/- 0.03 for the mice per genotype; t-test, p > 0.05) Figure Loss of PKCδ does not affect the number of actively dividing cells in uninjured taste epithelia A: PKCδ is detected in non-taste epithelium (NT) and circumvallate papillae by western blot B, C: Ki - 67 - IR (red) and sytox (green): Ki - 67 immunoreactive cells reside primarily in basal taste epithelium in WT (B) and PKCδ null (KO) mice (C) Ki - 67 labeling indices for WT and PKCδ null mice are not different (D; mean +/- SD, t-test, n = 3, p > 0.05) White asterisks in B, C indicate taste buds We next evaluated whether the number of circumvallate papilla (Fig 2A, B) Upon quanti- taste cells within taste buds were comparable fication, we found that the mean number of between PKCδ null mice and wild type mice, gustducin-IR cells per circumvallate papilla did using one specific taste cell marker: gustducin not differ between genotypes There was an immunoreactivity (IR) Gustducin is expressed average of 18.2 +/- 3.1 gustducin-IR taste strongly in the cytoplasm of a subset of type II cells for wild type mice, compared with an av- cells thought to transduce bitter taste Mutant erage of 17.6 +/- 2.1 gustducin-IR taste cells mice appeared to have comparable numbers for PKCδ null mice (Figure 2C; n=3 mice, of gustducin-IR cells within taste buds of the mean +/- SD, t-test, p > 0.05) Figure Type II cells are normal in non-irradiated PKCδ null mice JMR 105 E1 (7) - 2016 69 JOURNAL OF MEDICAL RESEARCH A, B: gustducin-ir (red) and sytox (green) Gustducin-ir cells are present in non-irradiated wild type mice (A) and in the circumvallate taste buds of PKCδ null mice (B) The number of gustducinIR cells in WT mice and in PKCδ null mice per circumvallate papilla were not significantly different (C; mean +/- SD, t-test, n = 3, p > 0.05) We next characterized the taste behaviors roughly 0.5 for both wild type and PKCδ null of mice using a two-bottle taste preference mice, indicating that neither genotype can test In this experiment, we used denatonium distinguish denatonium from water at these benzoate (which contains a strongly bitter low concentrations of bitter taste However, taste) to test avoidance behaviors exhibited by both wild type and PKCδ null mice avoided both wild type and PKCδ null mice Wild type denatonium at mM and mM (Figure 3; mice normally avoid drinking denatonium ben- way ANOVA, p > 0.05), indicating that there zoate at concentrations of 0.3 – mM Simi- was no difference in bitter taste sensitivity as larly, we found that at 0.03 mM and 0.1 mM assayed behaviorally for the wild type and denatonium, PKCδ null mice included in this study the preference ratios were Figure Prior to irradiation, behavior preferences for PKCδ null mice with regards to bitter taste are comparable to the preferences of wild type mice Mean (± SEM) preference ratios in 48 hours for the two - bottle tests with water and denatonium at 0.03 mM, 0.1 mM, mM and mM n = 3, way ANOVA, p > 0.05 To assess how irradiation affects actively dividing cells in the taste epithelium of PKCδ null mice, we irradiated PKCδ null mice, quantified proliferation indices in taste epithelia at dpi, dpi, dpi, and dpi and compared these values to those of non - irradiated controls and irradiated wild type mice In wild type mice, the labeling index for Ki - 67 was significantly reduced at and dpi, in comparison to the non - irradiated controls 70 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH Figure Mitigation of irradiation effects on proliferative cells of taste epithelium in PKCδ KO mice In WT mice, the Ki-67 labeling index was significantly decreased at and dpi and recovered to control levels at dpi In PKCδ null mice, the of In PKCδ null mice, the reduction in the labeling index for Ki-67 at and dpi was sig- proliferative cells was mitigated Significant nificantly less diminished compared to wild differences the type mice (Figure 4, n = - mice for each labeling indices of WT and PKCδ null mice time, way ANOVA, p < 0.05) Significant dif- after irradiation (black asterisks in G) Sytox ferences between wild type and PKCδ null (green) and Ki-67 (red) prior to irradiation, mice were detected at dpi and dpi (Tukey dpi, and dpi in wild type (A, B, C) and PKCδ test, p < 0.05) These results indicate that null mice (D, E, F) G: Ki - 67 labeling indices PKCδ is required for reduction of cell prolifera- of WT (yellow line) and PKCδ null mice (blue tion in the first few days following irradiation to line) under control conditions and following the head and neck detected JMR 105 E1 (7) - 2016 irradiation SD, way ANOVA, p < 0.05 on were effects irradiation N = - for each point Mean +/- between 71 JOURNAL OF MEDICAL RESEARCH IV DISCUSSION non-irradiated taste epithelia, PKCδ is in its inactive form and thus its loss causes no gross PKCδ null mice possess normal taste epithelia and exhibit normal taste behavior PKCδ plays a critical role in inducing apoptosis in response to insult; however, loss of this gene appears to have little consequence for mice when under normal homeostatic conditions For example, salivary epithelia are normal in uninjured PKCδ null mice [4] We found that taste epithelia in non - irradiated PKCδ null mice possess normal characteristics, and are no different in terms of several general measures than the taste epithelia in wild type mice PKCδ is expressed in both non -taste lingual epithelium and circumvallate papilla epithelium, consistent with reports that PKCδ is expressed in most tissues, including in mouse epidermal keratinocytes [5] Although PKCδ is expressed in taste epithelium, loss of PKCδ does not change the morphology of taste papillae and taste buds: these are indistinguishable in PKCδ null and wild type mice In PKCδ null mice, actively dividing cells are located in basal epithelium and perigemmal edge cells around taste buds, as is the case in wild type mice The proliferation index of the taste epithelium in PKCδ mice is also no phenotype; furthermore, this may suggest that PKCδ is only activated following stressful stimuli Loss of PKCδ protects taste progenitor proliferative activity in irradiated epithelium, suggesting potential therapeutic treatment by PKCδ antagonists Several studies have reported a role for PKCδ in inducing apoptosis and/or cell cycle arrest following injury by irradiation [3; 4; 7] Here we observed mitigation of the reduction in the proliferation index of the taste epithelium in PKCδ null mice after irradiation There are several possibilities as to how PKCδ may function in damaged taste tissues Disruption of PKCδ normally prevents taste progenitor cells from entering cell cycle arrest, which is thought to be necessary in order for cells to undergo DNA repair following irradiation damage In this case, PKCδ may negatively regulate proliferation, so that in the cells in the PKCδ null mice continue to divide Considering that cells use checkpoint systems to maintain the integrity of the genome during DNA replication [12], loss of PKCδ may destroy the genomic stability of the taste pro- different than that of wild type mice (Figure 1) genitor pool Thus, its effects on taste epithe- Finally, gustducin-IR type II cells are found in lium may actually be detrimental, and may normal numbers in mutant taste buds appear long after radiation treatment Another PKCδ null mice also possess normal taste possible theory is that PKCδ may be required avoidance with regards to the extremely bitter for apoptosis in taste progenitors after irradia- substance denatonium benzoate Wild type tion, as in the salivary gland In the PKCδ null mice cannot detect low concentration of dena- mice, then, we predict that the cell death that tonium, and start to avoid denatonium at 0.3 – normally occurs maximally in the first 24 hours mM [11] We found similar avoidance of de- following natonium at mM and mM for both PKCδ reduced To test these possibilities in future null mice and the wild type controls Our experiments, we might consider monitoring results apoptosis in PKCδ null mice during the first 24 72 suggest the possibility that in radiation treatment should be JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH hours after radiation If PKCδ is required for the loss of PKCδ would allow cells with un- taste progenitor cell death, cell death will be repaired DNA damage to progress through the reduced in the knockout mice We can also cell cycle, potentially leading to genomic insta- use immunomarkers to reveal the expression bility and the development of new cancerous of checkpoint proteins, including cdk1, cdk2 cells Nonetheless, understanding PKCδ func- cip1 and p21 The phosphorylation of cdk1 at tion might help us to better understand how tyr15 is critical for G2/M cell cycle arrest, while taste progenitors behave after irradiation and Cdk2 is linked to G1/S cell cycle arrest [13] which mechanisms are fundamental to enable cip1 P21 is downstream of PKCδ and is known taste bud cells to continuously be renewed to maintain G2/M arrest in UV-irradiated cells Acknowledgement [4] If PKCδ is involved in cell cycle arrest following irradiation, we suspect that cdk1, cdk2 and p21cip1 will be unregulated in taste proge- We would like to thank the staff that participated in this research nitors in wildtype irradiated epithelium, but not REFERENCES so in PKCδ mutants Recent studies have revealed the possibil- Verma A.K., Wheeler D.L., Aziz M.H et ity of using PKCδ inhibitors for therapeutic al (2006) Protein kinase Cepsilon and devel- treatment in a number of contexts Phase 2b opment of squamous cell carcinoma, the non- clinical trials are now underway to test the melanoma human skin cancer Mol Carcinog, safety and efficacy of a peptide PKCδ inhibitor 45, 381– 388 in reducing ischemia and reperfusion injury Yoshida K., Miki Y., Kufe D (2002) following acute myocardial infarction in hu- Activation of SAPK/JNK signaling by protein mans, as an adjunct to current treatments kinase Cdelta in response to DNA damage J Rottlerin, a PKCδ inhibitor, has been found to Biol Chem, 277, 48372 - 483728 protect against neuronal loss in both cell cul- LaGory E.L., Sitailo L.A., Denning M.F tures and preclinical animal models with Park- (2010) The protein kinase Cdelta catalytic inson’s disease, suggesting a potential thera- fragment is critical for maintenance of the G2/ peutic strategy for the treatment of Parkinson’s M DNA damage checkpoint J Biol Chem, 285, [14] To identify a PKCδ inhibitor that could 1879 - 1887 protect oral tissues from irradiation-induced Humphries M.J., Limesand K.H., damage, we must address several questions Schneider J.C et al (2006) Suppression of about the function of PKCδ in taste epithelium apoptosis in the protein kinase Cdelta null Does PKCδ induce cell cycle arrest and/or is it mouse in vivo J Biol Chem, 281, 9728 - 9737 required for apoptosis following irradiation? 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