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

Our initial results showed that the dramatic reduction in the proliferative index typically observed in the taste epithelia of irradiated wild type mice 1 - 3 days after irradiation was

MITIGATION OF IRRADIATION EFFECTS ON TASTE EPITHELIUM

IN THE PROTEIN KINASE C DELTA NULL MOUSE

Nguyen Manh Ha 1 , Nguyen Xuan Hoi 2

1 Hanoi Medical University, 2 National 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 1 - 3 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

Corresponding author: Nguyen Xuan Hoi, National

Obstet-rics and Gynecology Hospital

E-mail: doctorhoi@gmail.com

Received: 20 October 2016

Accepted: 10 December 2016

I INTRODUCTION

Protein kinase C (PKC) belongs to a large

serine/threonine protein kinases The many

isoforms of PKC have been categorized into 3

subfamilies: classical (α, βI, βII, and γ), novel

(δ, ε, η, and θ) and atypical (λ and ζ) The

different isoforms of PKC play multifaceted

roles in cellular response in a range of tissues

Among the many isoforms of PKC, at least five

(α, δ, ε, η, and ζ) are expressed in epidermal

keratinocytes [1]; in fact, PKCδ was originally

discovered in the mouse epidermis Upon

activation, PKCδ has been shown to be a key

regulator of the intrinsic

(mitochondrial-dependent) apoptotic pathway

PKCδ is activated by a variety of stimuli, including ionizing radiation, anti-cancer drugs, oxidative stress, ultraviolet radiation, and organelle poisons [2] PKCδ promotes apoptosis by activating many proteins in the apoptotic cascade, including p53, caspase 9, caspase 3, caspase 7, Mcl-1 and lamin B Some studies have shown that PKCδ also functions in cell cycle regulation The PKCδ catalytic fragment has a critical role in enforcing the G2/M checkpoint in response to

UV radiation in human keratinocytes [3] 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

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],

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

reperfu-sion 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

hypothe-sized 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 do

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

II SUBJECTS AND METHODS

1 Animals

Two- to four-month-old C57Bl/6 and PKCδ-/

-mice were used in all experiments

2 Methods

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 immunofluores-cence, 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 2 hours at room temperature, then soaked for 15 minutes each with avidin/biotin blocking solutions (A/B blocking kit; Vector Laborato-ries) Sections were then incubated in rabbit anti - Ki - 67 antiserum (1: 200; Thermo Scien-tific) 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 Inter-national) in PBS buffer for two hours at room temperature After a final wash in PBS for one hour, sections were counter-stained with Sytox green (Invitrogen) and mounted in Fluoro-mount G (SouthernBiotech)

Isolation of lingual epithelium for west-ern blot analysis of PKCδ

The method that we used was adapted

from Behe et al and Vandenbeuch et al [9]

Adult mice were killed by CO2inhalation and

cervical dislocation Tongues from the wild

type mice were quickly removed and stored for

several minutes in ice-cold Tyrode’s solution

equilibrated with 100% oxygen Tyrode’s

solu-tion contains 140 nM NaCl, 5 nM KCl, 1 nM

MgCl2, 2 nM CaCl2, 10 nM HEPES, 10 nM

glucose, and 1 nM Na pyruvate, pH 7.4

Lin-gual epithelium containing taste buds were

pealed off of the tongue’s muscle, rinsed in

Ca2+/Mg2+-free Tyrode’s, and stored at -800C

Immunoblot for PKCδ

The method for western immunoblot for

PKCδ has been described elsewhere [4]

Briefly, the circumvallate papillae and

non-taste epithelium were taken from three wild

type mice Protein was suspended in JNK lysis

buffer and Rabbit polyclonal antibody (Santa

Cruz) was used to detect PKCδ in a 1: 1000

diluted solution

Two-bottle preference test

Preferences for water versus different

con-centrations of denatonium (which contains a

bitter taste) were tested in wild type and PKCδ

null mice using conventional methods [10]

Two tubes were used, one which contained

either water or denatonium at different

concentrations (tube 1) and the other which

contained water (tube 2) The mice were given

48-hour access to two drinking tubes The

positions of the tubes were randomized and

switched after 24 hours to avoid place

prefe-rences Consumption of denatonium solution

and water was recorded for the entire 48-hour

period The following concentrations of

dena-tonium were used: 0.03 mM; 0.1 mM; 1 mM;

and 3 mM Preference scores were defined as ratios of the total solution consumed in tube 1 divided by the total solution consumed in tubes 1 and 2

Analysis

To analyze the data, we obtained images, counted cells, and calculated the proliferation index using Ki-67-ir The data for the two bottle taste preference test was gathered from three C57Bl/6 mice and three PKCδ null mice For the immunoblot, epithelia were collected from three C57Bl/6 and PKCδ null mice Data

collected from three to four mice per time point All cell counts were corrected with the Abercrombie correction factor to account for any variability in cell size following irradiation treatment A T-test or 2 way ANOVA was used

to analyze data

III RESULTS

Characterization of taste epithelium in PKCδ mice

To examine whether PKCδ was expressed

in the lingual epithelium of the wild type mice,

we performed an immunoblot of the lingual epithelium to detect the PKCδ protein We found that PKCδ was present in both the non-taste epithelium (NT) and the non-taste epithelium

of the circumvallate papilla (CV; Figure 1A) Next, to assess whether the proliferative activ-ity of taste epithelium in PKCδ null mice was comparable to that of wild type mice, we employed Ki-67-IR, which marks actively proliferating cells in all phases of the cell cycle except for early G1 and G0 In PKCδ null mice, actively dividing cells were detected in the basal epithelium and in perigemmal

re-gions around taste profiles (Figure 1C), similar

to the pattern in wild type mice (Figure 1B)

The proliferation indices of Ki-67 for basal

taste epithelial cells were 0.79 +/- 0.03 for the

wild type mice and 0.81 +/- 0.02 for the PKCδ null mice These indices were not significantly different (Figure 1D: mean +/- SD, n = 3 - 4 mice per genotype; t-test, p > 0.05)

Figure 1 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

taste cells within taste buds were comparable

between PKCδ null mice and wild type mice,

using one specific taste cell marker: gustducin

immunoreactivity (IR) Gustducin is expressed

strongly in the cytoplasm of a subset of type II

cells thought to transduce bitter taste Mutant

mice appeared to have comparable numbers

of gustducin-IR cells within taste buds of the

circumvallate papilla (Fig 2A, B) Upon quanti-fication, we found that the mean number of gustducin-IR cells per circumvallate papilla did not differ between genotypes There was an average of 18.2 +/- 3.1 gustducin-IR taste cells for wild type mice, compared with an av-erage of 17.6 +/- 2.1 gustducin-IR taste cells for PKCδ null mice (Figure 2C; n=3 mice, mean +/- SD, t-test, p > 0.05)

Figure 2 Type II cells are normal in non-irradiated PKCδ null mice

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

gustducin-IR 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

of mice using a two-bottle taste preference

test In this experiment, we used denatonium

benzoate (which contains a strongly bitter

taste) to test avoidance behaviors exhibited by

both wild type and PKCδ null mice Wild type

mice normally avoid drinking denatonium

ben-zoate at concentrations of 0.3 – 3 mM

Simi-larly, we found that at 0.03 mM and 0.1 mM

denatonium, the preference ratios were

roughly 0.5 for both wild type and PKCδ null mice, indicating that neither genotype can distinguish denatonium from water at these low concentrations of bitter taste However, both wild type and PKCδ null mice avoided denatonium at 1 mM and 3 mM (Figure 3; 2 way ANOVA, p > 0.05), indicating that there was no difference in bitter taste sensitivity as assayed behaviorally for the wild type and PKCδ null mice included in this study

Figure 3 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, 1 mM and 3 mM n = 3, 2 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 1 dpi, 3 dpi,

5 dpi, and 7 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 1 and

3 dpi, in comparison to the non - irradiated controls

Figure 4 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 1 and 3 dpi and

recovered to control levels at 5 dpi In PKCδ

null mice, the effects of irradiation on

proliferative cells was mitigated Significant

differences were detected between the

labeling indices of WT and PKCδ null mice

after irradiation (black asterisks in G) Sytox

(green) and Ki-67 (red) prior to irradiation, 3

dpi, and 7 dpi in wild type (A, B, C) and PKCδ

null mice (D, E, F) G: Ki - 67 labeling indices

of WT (yellow line) and PKCδ null mice (blue

line) under control conditions and following

irradiation N = 3 - 4 for each point Mean

+/-SD, 2 way ANOVA, p < 0.05

In PKCδ null mice, the reduction in the la-beling index for Ki-67 at 1 and 3 dpi was sig-nificantly less diminished compared to wild type mice (Figure 4, n = 3 - 4 mice for each time, 2 way ANOVA, p < 0.05) Significant dif-ferences between wild type and PKCδ null mice were detected at 1 dpi and 3 dpi (Tukey test, p < 0.05) These results indicate that PKCδ is required for reduction of cell prolifera-tion in the first few days following irradiaprolifera-tion to the head and neck

non-irradiated taste epithelia, PKCδ is in its inactive form and thus its loss causes no gross phenotype; furthermore, this may suggest that PKCδ is only activated following stressful stimuli

Loss of PKCδ protects taste progenitor proliferative activity in irradiated epithe-lium, 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

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-genitor pool Thus, its effects on taste epithe-lium may actually be detrimental, and may appear long after radiation treatment Another possible theory is that PKCδ may be required for apoptosis in taste progenitors after irradia-tion, as in the salivary gland In the PKCδ null mice, then, we predict that the cell death that normally occurs maximally in the first 24 hours following radiation treatment should be reduced To test these possibilities in future experiments, we might consider monitoring apoptosis in PKCδ null mice during the first 24

IV DISCUSSION

PKCδ null mice possess normal taste

epithelia and exhibit normal taste behavior

PKCδ plays a critical role in inducing

apop-tosis 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

characteris-tics, 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]

Al-though PKCδ is expressed in taste epithelium,

loss of PKCδ does not change the morphology

of taste papillae and taste buds: these are

in-distinguishable in PKCδ null and wild type

mice In PKCδ null mice, actively dividing cells

are located in basal epithelium and

perigem-mal edge cells around taste buds, as is the

case in wild type mice The proliferation index

ofthe taste epithelium in PKCδ mice is also no

different than that of wild type mice (Figure 1)

Finally, gustducin-IR type II cells are found in

normal numbers in mutant taste buds

PKCδ null mice also possess normal taste

avoidance with regards to the extremely bitter

substance denatonium benzoate Wild type

mice cannot detect low concentration of

dena-tonium, and start to avoid denatonium at 0.3 –

3 mM [11] We found similar avoidance of

de-natonium at 1 mM and 3 mM for both PKCδ

null mice and the wild type controls Our

results suggest the possibility that in

hours after radiation If PKCδ is required for

taste progenitor cell death, cell death will be

reduced in the knockout mice We can also

use immunomarkers to reveal the expression

of checkpoint proteins, including cdk1, cdk2

and p21cip1 The phosphorylation of cdk1 at

tyr15 is critical for G2/M cell cycle arrest, while

Cdk2 is linked to G1/S cell cycle arrest [13]

P21cip1is downstream of PKCδ and is known

to maintain G2/M arrest in UV-irradiated cells

[4] If PKCδ is involved in cell cycle arrest

fol-lowing irradiation, we suspect that cdk1, cdk2

and p21cip1 will be unregulated in taste

proge-nitors in wildtype irradiated epithelium, but not

so in PKCδ mutants

Recent studies have revealed the

possibil-ity of using PKCδ inhibitors for therapeutic

treatment in a number of contexts Phase 2b

clinical trials are now underway to test the

safety and efficacy of a peptide PKCδ inhibitor

in reducing ischemia and reperfusion injury

following acute myocardial infarction in

hu-mans, as an adjunct to current treatments

Rottlerin, a PKCδ inhibitor, has been found to

protect against neuronal loss in both cell

cul-tures and preclinical animal models with

Park-inson’s disease, suggesting a potential

thera-peutic strategy for the treatment of Parkinson’s

[14] To identify a PKCδ inhibitor that could

protect oral tissues from irradiation-induced

damage, we must address several questions

about the function of PKCδ in taste epithelium

Does PKCδ induce cell cycle arrest and/or is it

required for apoptosis following irradiation? If

loss of PKCδ protects taste progenitors from

apoptosis without serious side effects on a

patient’s heath, we might consider PKCδ

in-hibitors as a potential therapy for head and

neck cancer patients undergoing irradiation

However, if PKCδ induces cell cycle arrest,

the loss of PKCδ would allow cells with un-repaired DNA damage to progress through the cell cycle, potentially leading to genomic insta-bility and the development of new cancerous cells Nonetheless, understanding PKCδ func-tion might help us to better understand how taste progenitors behave after irradiation and which mechanisms are fundamental to enable taste bud cells to continuously be renewed

Acknowledgement

We would like to thank the staff that partici-pated in this research

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