Morphometric evaluation of the turnover of autophagic vacuoles after treatment with Triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells

Large numbers of autophagic vacuoles were found in murine pancreatic acinar and seminal vesicle epithelial cells following the administration of Triton X-100 or vinblastine for 4 h. The autophagic vacuoles disappeared rapidly from the cells after the administration of cycloheximide to animals pretreated with Triton X-100. The decay in seminal vesicle cells appeared to follow first-order kinetics with an estimated t1/2 of 8.7 min. The regression in pancreatic cells was equally rapid and less than half the initial volume of autophagic vacuoles was found at the 12th min after cycloheximide injection. This time, the decay curve appeared to be linear rather than exponential. Our data, together with the work of others, support the view that the average half-life of autophagic vacuoles is a fairly constant parameter kept within the range of 6-9 min in various types of mouse and rat cell when the late steps of autophagocytosis (i.e. the fusion of autophagosomes and lysosomes and the degradation within lysosomes) are not affected. The regression of autophagic vacuoles was slow in mice pretreated with vinblastine (t1/2 of about 27-30 min) suggesting that this drug slows down the turnover of autophagic vacuoles. Morphometric evaluation of the regression of the autophagic vacuole compartment after cycloheximide treatment can be used as a tool to distinguish between treatments which elevate the amount of autophagic vacuoles within the cells by increasing the rate of sequestration from those which expand the autophagic vacuole compartment by causing accumulation of autophagic vacuoles as a result of blockade of the late steps of the autophagic process.     

Effect of different protein synthesis inhibitors on the exocrine pancreatocytic autophagocytosis in vivo.

The translational inhibitor cycloheximide is also used as an inhibitor of cellular autophagy and intracellular degradation of endogenous cellular proteins. Some evidence for a similar effect of other inhibitors of protein biosynthesis is also available (largely from in vitro systems). In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the fact that when the formation of autophagosomes is inhibited, a regression of the autophagolysosomal compartment can be measured, provided intralysosomal degradation in the pre-existing autophagic vacuoles continues at an unchanged rate. To make the measurements easier, autophagolysosomal compartment of the cells was enlarged by administering vinblastine (10 mg/kg b.wt.) for 2 h when the inhibitors were given for an additional 30 min. During this time cycloheximide (0.2 mg/g b.wt.), emetine (0.12 mg/g b.wt.) and puromycin (0.2 mg/g b.wt.), respectively caused 35, 25 and 52% regression of the pancreatocytic autophagolysosomal compartment. Since all the above translational inhibitors inhibited autophagocytosis as well, the possibility of a coupling between the regulation of synthesis and inhibition of proteins arises.     

Regression of autophagic vacuoles in mouse pancreatic cells: a morphometric study of the effect of methylamine and chloroquine followed by cycloheximide treatment

Treatment of mice with methylamine and chloroquine for two hours markedly increased the volume fraction of autophagic vacuoles in the pancreatic acinar cells of the mouse. The autophagic vacuoles disappeared from the cells within 20 min after the administration of a suppressor of autophagic sequestration cycloheximide to animals pretreated with 0.25 mg b.w. dose of methylamine. In contrast, no regression or very slow decay was seen in cells of mice pretreated with higher doses of methylamine (0.50-0.70 mg/g b.w.) and chloroquine (0.08 mg/g b.w.). Our data show that the two drugs retard the disintegration of autophagic vacuole content. It is concluded, that accumulation of autophagic vacuoles due to their slow turnover is an important mechanism of the expansion of autophagic vacuole compartment under the effect of methylamine and chloroquine.     

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.     

Changes in the extents of viable and necrotic tissue, interstitial fluid pressure, and proliferation kinetics in clone A human colon tumour xenografts as a function of tumour size

Abstract. Total, viable and necrotic tumour tissue, tumour cell yields, and colony forming efficiencies were measured in clone A human colon tumour xenografts as neoplasms grew from about 100 mm³ to about 6000 mm³. The volumes of the total, viable and necrotic compartments were fit using the Verhulst equation to obtain estimates of growth rates and maximal sizes of the various compartments (carrying capacities). Additionally, at four discrete tumour volumes (250, 850, 2500 and 5500 mm³), hypoxic percentages, proportions of parenchymal tumour and host cells, interstitial fluid pressures, and proliferation kinetics including measurements of apoptosis were determined. There were interesting relationships between the shapes of the curves for total, viable and necrotic tissue to some of the other endpoints measured. Specifically, the volumetric growth curves for the total and viable tumour tissue compartments were identical to a volume of approximately 1000 mm³, but diverged at larger sizes, with the viable cell compartment exhibiting a smaller carrying capacity. The shape of the growth curve for the necrotic compartment exactly mimicked that for the total volume compartment, but was delayed in time by about 21 days. Similarity in shape to that of the overall tumour volume/necrotic volume curves was also seen for the curve for the increase in interstitial fluid pressure, and for the increase in the size of the host cell compartment. In contrast, the growth of the hypoxic compartment and of the parenchymal tumour cell compartment were similar in shape to that of the viable compartment. These data indicate that these compartments are functionally linked. Marked changes in cell kinetic parameters occurred as tumour size increased from 250–5500 mm³. The labelling index and growth fractions decreased from 0.256–0.125, and 0.77–0.40 respectively, and the cell loss factor increased from 0.52–0.74. The volumetric and potential doubling times increased from 4.3–17.6 and 2.1–4.6 days respectively. The cell kinetic changes could not be clearly related to the changes in shape of either the overall tumour volume or the viable tumour volume.     

Time course of vinblastine-induced cellular autophagy in the murine pancreatic acinar cells in vivo. Different regression rates of the autophagic compartment caused by cycloheximide given different times after the vinca alkaloid. A morphometric study.

Autophagy is a three-step process in which parts of cytoplasm are segregated by membranes to form autophagosomes gaining acid hydrolases later, being converted this way into autolysosomes in which lysosomal degradation takes place. The actual size of the autophagic vacuole compartment (AVC) is obviously dependent on the velocity of these main steps. According to our morphometric measurements, a single dose (10 mg/kg b.wt.) of vinblastine (VBL) caused a conspicuous expansion of the AVC in pancreatic acinar cells, occurring in two waves: it expanded in the first 90 min but regressed in the next hour. This was followed by a second expansion monitored until the 5th post-injectional hour. The expansion rates indicate the existence of stimulation of autophagic segregation in both expansion phases. To take a further look, into the dynamics of the process, we blocked segregation by giving cycloheximide (CHI 0.2 mg/g b.wt.) 1 and 3 h after VBL and the subsequent regression of the AVC was followed by morphometry in the next 90 min. At the height of the first wave (1-2 h after VBL) the regression of AVC was not retarded, but rather, degradation rate seemed elevated. When CHI was given 1 h after VBL, 92% of the cytoplasmic volume fraction (CVF) of AVC regressed within the next 30 min. The main factor causing the expansion of AVC might be enhanced segregation in the first wave. Contrarily, at the beginning of the second wave, the turnover of AVs is dramatically slowed down. When CHI was given 3 h after VBL, only 27% of CVF of AVC regressed in the next 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Daunomycin and Daunomycin-protein conjugates on the ultrastructure of Trypanosoma rhodesiense

The nucleus (but not the kinetoplast) of Trypanosoma rhodesiense is the principal target for trypanocidal attack by Daunomycin in vitro and Daunomycin-protein conjugates in vivo; free Daunomycin is inactive in vivo. Nucleolar segregation and abnormal distribution of heterochromatin occur after drug treatment as in tumour cells. Other abnormal changes include formation of cytoplasmic clefts and autophagic vacuoles, increased prominence of the tubular collection system, and exocytotic vesicle production. Large masses of ferritin accumulate in white cells after treatment with the drug-ferritin conjugate but no ferritin was detectable in trypanosomes.     

Characterisation of the progression of azaserine-induced rat pancreatic adenocarcinoma by proliferative cell nuclear antigen,basement membrane laminin and trypsinogen immunohistochemistry

The progression of azaserine-induced rat pancreatic adenocarcinoma (AC) was characterised using quantitative and semiquantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA), basement membrane laminin (BML) and trypsinogen (TG). Samples were taken 5-20 months after initiation. High PCNA-labelling indices (PCNA LIs) were measured 5 months after the induction of atypical acinar cell nodules (AACNs), which decreased later and stagnated until a further decline in the month 10 adenomas. Then a second premalignant proliferative wave was observed (month 13) within the adenoma stage. Later, in month 20 differentiated ACs PCNA LIs fell to the host tissue level but were found highest in the month 20 anaplastic ACs indicating a switch to malignant proliferation. Month 20 invasive ACs showed a number of separate proliferative foci. In early AACNs, BML decreased and remained low till the local maximum in the month 13 adenoma. Invasive ACs did not express BML. Month 5 AACN and differentiated AC were TG deficient but anaplastic AC regained its TG expression. However invasive AC was again TG negative. These results are discussed in combination with our previous data on progressional changes of autophagic capacity and microvessel densities.     

Protein degradation in lysosomes from chemically induced malignant rat hepatoma

Hepatocellular carcinomas were induced in rat liver by exposing the animals to diethylnitrosamine and 2-acetylaminofluorene in combination with partial hepatectomy. Light and electron microscopy demonstrated that the general appearance of the tumour tissue was that of highly differentiated malignant hepatocytic cells. Morphometrically there was a difference between normal and malignant cells in that the entire lysosomal apparatus was twice as large in malignant cells as in normal cells. This was mainly due to an increase in the fractional volume of autophagic vacuoles. A total lysosomal fraction (dense bodies and autophagic vacuoles) was isolated and characterized from both control and tumour livers. Marker enzyme analysis showed that the lysosomal enzyme activities were significantly lower in malignant liver tissue. Injection of leupeptin, an inhibitor of cathepsins B, H, and L, into rats did not increase the fractional volume of autophagic vacuoles in tumour tissue as much as in normal liver tissue. The proteolytic rate was lower in the lysosomal fraction from hepatoma cell tissue compared with the lysosomal fraction from normal cell tissue. This could conceivably be due to the lower activities of lysosomal enzymes. However, if the recovery of lysosomes is taken into account no clear-cut difference in lysosomal proteolysis between control and malignant liver tissue was noted. Accordingly, in malignant liver tissue a proteolytic balance is obtained characterized by an increased fractional volume of AVs and lower rate of protein degradation in individual lysosomes.     

Alteration with age of symbiosis of gene expression in aphid endosymbionts

Aphid endosymbionts in vivo in young hosts synthesized almost exclusively only one protein, symbionin. The synthesis of symbionin declined with age of the host and instead the endosymbiont began to express some of its own genes which were expressed in vitro but were repressed in vivo in young host. A prolonged treatment of young host with cycloheximide brought about a physiological state similar to that in old insect. Though in the very old insect symbionin was no longer produced by its endosymbiont, the host seemed to depend almost entirely upon the gene products of the endosymbiont.     

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.     

Lutropin increases phosphorylation of a 33 000-dalton ribosomal protein in rat tumour Leydig cells.

Addition of lutropin (luteinizing hormone, 'LH') and 3-isobutyl-1-methylxanthine to tumour Leydig cells stimulated phosphorylation of five proteins, of 17 000, 22 000, 24 000, 33 000 and 57 000 Da. Phosphorylation of these proteins coincided with increased pregnenolone production. Phosphorylation of a 33 000-Da protein was lutropin-dependent in Leydig cells isolated from a Leydig-cell tumour, from immature testes or from mature testes. In tumour Leydig cells this protein was present in the small ribosomal subunit. Incubation of tumour Leydig cells with either cycloheximide or puromycin inhibited both basal and lutropin-dependent pregnenolone production, by approx. 90% and 98% respectively. In contrast, basal pregnenolone production in Leydig cells from immature and mature testes was insensitive to cycloheximide or puromycin. Cycloheximide or puromycin increased phosphorylation of the 33 000-Da phosphoprotein by approx. 130% and 80% respectively (effect of lutropin/3-isobutyl-1-methylxanthine on phosphorylation: 100%). The molecular mass, the subcellular localization and the sensitivity to phosphorylation in the presence of inhibitors of protein synthesis indicate that the 33 000-Da protein could be similar to ribosomal protein S6.     

Differentiation-dependent expression of cathepsin D and importance of lysosomal proteolysis in the degradation of UCP1 in brown adipocytes

The lysosomal protease cathepsin D increased markedly in brown adipocytes during differentiation in primary cultures. Differentiated cells had 20 times the amount of immunoreactive cathepsin D found in preadipocytes. Cathepsin D mRNA, as estimated by relative RT-PCR, was also present in higher amounts in differentiated brown fat cells. Cathepsin D expression was not influenced by repeated exposures of brown adipocytes to norepinephrine (NE). Cathepsin D levels were also unchanged when NE was withdrawn for 48 h after cells had been exposed to NE for 7 days. In contrast, exposure of the cells to NE for 7 days increased their UCP1 content by more than twofold, which returned to basal levels within 48 h of withholding NE. The half-life of UCP1 under basal conditions and in cells chronically exposed to NE was estimated from reductions in [35S]methionine-labelled immunoprecipitable UCP1 over 72 h. UCP1 t1/2 under basal conditions was 3.7+/-0.4 days, which was similar to the half-lives of labelled mitochondrial translation products (3.6+/-0.8 days). The turnover rates of both UCP1 and mitochondrial translation products were reduced by NE. The turnover rate of UCP1 in the presence or absence of NE cannot account solely for the rapid loss of UCP1 from brown adipocytes upon withdrawal of NE. This loss was reduced when cells were incubated with inhibitors of phosphatidylinositol 3-kinases (PI 3-kinase), previously shown to block formation of autophagic vacuoles. Thus, brown adipocytes acquire a large capacity for both uncoupled metabolism and for lysosomal proteolysis during differentiation. Withdrawal of NE, as often occurs in vivo from suppression of sympathetic nervous system activity, would not only terminate thermogenesis but also favor formation of autophagic vacuoles to rapidly reduce the cell content of UCP1-containing mitochondria.     

Occurrence and inhibition by cycloheximide of apoptosis in vinblastine-treated murine pancreas. A role for autophagy?

While investigating the time course of the influence of autophagy-inducer vinblastine in pancreatic acinar cells, we discovered that about 24 h after the single injection of the drug (10 mg/kg b.wt.) when most of the cells were already recovering from the autophagic wave, dying cells with apoptotic nuclei and peculiar morphology of their cytoplasm appeared in the exocrine pancreas. Apoptotic blebs (ABs) separating from apoptotic cells or already phagocytized by neighbouring cells were also also observed. A large number of autophagic vacuoles (AVs) were seen in these cells termed apoptotic cell (AC). They are most probably derived from cells with morphologically normal nuclei but with unusually high number of AVs in their cytoplasm. We termed these cells highly autophagic cells (HAC). Our morphometric measurements show that the partial volume of both HAC and AC increased to 7.5 and 9.5%, respectively, of total cellular volume in the 25th h after vinblastine treatment. This increase could be inhibited by a treatment at the 24th h with cycloheximide (0.2 mg/g b.wt.) an inhibitor of both translation and autophagic segregation. Thus, synthesis of proteins or an enhanced autophagy may be indispensable step(s) in the apoptotic process in this system.     

Inhibition by insulin of the physiological autophagic breakdown of cell organelles

By using electron microscopic morphometry it was found that the fractional volume of autophagic vacuoles in the cytoplasm of liver cells decreased rapidly after administering 5 units insulin per kg body weight to otherwise untreated rats. From the decay which appears to follow first order kinetics a half-life of 9 min was found for the compartment of autophagic vacuoles. The rates of autophagic breakdown calculated were different for the different cytoplasmic components, indicating that selecting mechanisms are involved in the process of segregation which is the first step in autophagy.     

Manipulation of autophagy by bacteria for their own benefit

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.     

Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner

Plant genomes harbor autophagy-related (ATG) genes that encode major components of the eukaryotic autophagic machinery. Autophagy in plants has been functionally linked to senescence, oxidative stress adaptation and the nutrient starvation response. In addition, plant autophagy has been assigned negative ('anti-death') and positive ('pro-death') regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. The role of autophagy in plant disease and basal immunity to microbial infection has, however, not been studied in detail. We have employed a series of autophagy-deficient genotypes of the genetic model plant Arabidopsis thaliana in various infection systems. Genotypes lacking ATG5, ATG10 or ATG18a develop spreading necrosis and enhanced disease susceptibility upon infection with toxin-producing pathogens preferring a necrotrophic lifestyle. These findings suggest that autophagy positively controls the containment of host tissue integrity upon infections by host-destructive microbes. In contrast, autophagy-deficient genotypes exhibit markedly increased immunity to infections by biotrophic pathogens through altered homeostasis of the plant hormone salicylic acid, thus suggesting an additional negative regulatory role of autophagy in plant basal immunity. In sum, our findings suggest that the role of plant autophagy in immunity cannot be generalized, and depends critically on the lifestyle and infection strategy of invading microbes.     

Thyrotropin-Releasing Hormone Release Is Enhanced in Hippocampal Slices After Electroconvulsive Shock

Abstract: Hippocampal thyrotropin-releasing hormone (TRH) release was examined after seizures were induced by electroconvulsive shock (ECS). Rat hippocampal slices taken 12, 24, or 48 h after 3 days of alternate-day ECS treatment or sham-ECS treatment were stimulated with potassium with or without calcium in a superfusion system containing in-line charcoal adsorbent to concentrate TRH. Released TRH and tissue TRH were measured by radioimmunoassay. The TRH content of hippocampal slices was increased fivefold over sham-ECS levels 12, 24, and 48 h after ECS, but this was not associated with an increase in basal TRH release. Potassium-stimulated TRH release was significantly elevated over basal release 12, 24, and 48 h after ECS. Potassium-stimulated calcium-dependent TRH release increased linearly after ECS, reaching its highest level 48 h after seizure. Thus, although enhanced calcium-dependent TRH release was associated with elevated tissue levels, this relationship was not proportional in that tissue TRH was elevated to the same extent at all times after ECS, whereas potassium-evoked calcium-dependent TRH release increased gradually over time after seizure. These results suggest that postictal elevations in TRH are associated with an enhanced capacity for release that develops as a result of a time-dependent shift of TRH from a storage compartment to a readily releasable pool. The observed elevation in stimulated TRH release may be relevant to seizure-induced modulation of TRH receptors in vivo.     

Treatment with HPMA copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice

Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox–PHPMA–HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10–20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8⁺ lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.     

mTOR Inhibition Prevents Epithelial Stem Cell Senescence and Protects from Radiation-Induced Mucositis

The integrity of the epidermis and mucosal epithelia is highly dependent on resident self-renewing stem cells, which makes them vulnerable to physical and chemical insults compromising the repopulating capacity of the epithelial stem cell compartment. This is frequently the case in cancer patients receiving radiation or chemotherapy, many of whom develop mucositis, a debilitating condition involving painful and deep mucosal ulcerations. Here, we show that inhibiting the mammalian target of rapamycin (mTOR) with rapamycin increases the clonogenic capacity of primary human oral keratinocytes and their resident self-renewing cells by preventing stem cell senescence. This protective effect of rapamycin is mediated by the increase in expression of?mitochondrial superoxide dismutase (MnSOD), and the consequent inhibition of ROS formation and oxidative stress. mTOR inhibition also protects from the loss of proliferative basal epithelial stem cells upon ionizing radiation in?vivo, thereby preserving the integrity of the oral mucosa and protecting from radiation-induced mucositis.