Mouse Tongue Epithelium Research Articles

Investigation of Roles of SLC38A1 in Proliferation and Differentiation of Mouse Tongue Epithelium and Expression in Human Oral Tongue Squamous Cell Carcinoma.

The aerobic glycolytic pathway, boosting lactate formation, and glutamine addiction are two hallmarks of cancer pathophysiology. Consistent with this, several cell membrane glutamine transporters, belonging to different solute carrier (SLC) families, have been shown to be upregulated in a cell-specific manner to furnish the cells with glutamine and glutamine-derived metabolic intermediates. Among them, the system A transporter Slc38a1 has a higher affinity for glutamine compared to other SLC transporters, and it undergoes highly multifaceted regulation at gene and protein levels. The current study aimed to investigate the functional role of Slc38a1 in the proliferation and maturation of the mouse tongue epithelium. Secondly, we aimed to examine the expression of SLC38A1 and its regulation in human tongue oral squamous cell carcinoma (OTSCC). Employing Slc38a1 wild-type and knockout mice, we showed that Slc38a1 was not directly linked to the regulation of the proliferation and differentiation of the mouse tongue epithelium. External transcriptomic datasets and Western blot analyses showed upregulation of SLC38A1 mRNA/protein in human OTSCC and oral cancer cell lines as compared to the corresponding controls. Further, an investigation of external datasets indicated that mechanisms other than the amplification of the SLC38A1 chromosomal locus or hypomethylation of the SLC38A1 promoter region might be important for the upregulation of SLC38A1 in OTSCC.

The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus

The IRE1α-XBP1s signaling branch of the unfolded protein response is a well-characterized survival pathway that allows cells to adapt to and resolve endoplasmic reticulum stress. Recent data has broadened our understanding of IRE1α-XBP1s signaling beyond a stress response and revealed a physiological mechanism required for the differentiation and maturation of a wide variety of cell types. Here we provide evidence that the IRE1α-XBP1s signaling pathway is required for the proliferation and maturation of basal keratinocytes in the mouse tongue and esophageal epithelium. Mice with conditional targeted deletion of either Ire1α or Xbp1 in keratin 14 expressing basal keratinocytes displayed severe thinning of the lingual and esophageal mucosa that rendered them unable to eat. In IRE1α null epithelium harvested at an earlier timepoint, genes regulating cell proliferation, cell-cell adhesion, and keratinization were significantly downregulated; indirect immunofluorescence revealed fewer proliferating basal keratinocytes, downregulation of E-cadherin, and thinning of the loricrin-positive granular and cornified layers. The number of Tp63-positive basal keratinocytes was reduced in the absence of IRE1α, and expression of the Wnt pathway transcription factor LEF1, which is required for the proliferation of lingual transit amplifying cells, was also significantly downregulated at the transcript and protein level. Together these results reveal an essential role for IRE1α-XBP1s in the maintenance of the stratified squamous epithelial tissue of the tongue and esophagus.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Abstract A31: Transcriptional addiction to YAP1—a major driving force of oral cancer carcinogenesis and evolution?

Abstract Background: In this era of precision medicine, a clinically effective molecular target of HNSCC has not been identified so far. This is mainly because the predominant genetic alterations in HNSCC are loss-of-function (LOF) mutations in tumor-suppressor genes (TSGs), which are generally undruggable targets. We hypothesized that certain effector molecules liberated from the TSGs restriction may play an essential role in HNSCC oncogenesis and evolution. In this context, we analyzed the oncogenic function of transcription coactivator YAP1, which is the essential downstream effector protein of the Hippo pathway. Experiments and Results: We first examined the effect of mouse tongue epithelium specific conditional double knockout (cDOK) of Mob1a/b, the core component of the Hippo pathway that inhibits YAP1, employing CreERT; Mob1aflox/flox; Mob1b−/− mouse. Consequent YAP1 activation (but not TAZ) developed extremely rapid and highly reproducible (100%) tongue tumors: carcinoma in situ (2 weeks) and invasive carcinoma (4 weeks). The tongue epithelium cells isolated from the cDOK mouse demonstrated YAP1-dependent aggressive phenotypes. In human oral HNSCC samples, expanding accumulation of nuclear YAP1 protein was observed from the basal stem/progenitor cells to invasive cancer cells, and this spreading overexpression was significantly correlated with poor prognosis of patients. The YAP1 target module expression, established thorough the microarray assays with cDKO mouse cells, also correlated with the unfavorable prognosis of patients in the TCGA data. To elucidate the epigenetic mechanism that underlies this extremely rapid carcinogenesis, we then conducted chip-seq for cDKO mouse cells with or without YAP1 activation. Our preliminary data indicated that YAP1-targeted genes predominantly reside in the euchromatin islands and, surprisingly, their enhancers are marked with active H3K27ac, even in the YAP1-off condition, thus ready to respond to YAP1 nuclear translocation and thereby to start transcription, contradicting our expectation that genome-wide chromatin remodeling must be required in this rapid carcinogenesis model. Given that the presumed physiologic roles of YAP1 are a monitor of microenvironment and an emitter of regenerative cue under emergent cellular stressors (e.g., tissue injury) in the normal stem/progenitor cells, it is plausible that YAP1-target genes are kept in the ready-to-go status and constitutive activation of YAP1 and subsequent expression of targeted genes lead to the rapid carcinogenesis, causing the condition of “wounds that never heal.” Finally, our data and a recent study by Prof. Gutkind’s laboratory indicate that the major LOF of TSGs (e.g., p53 and FAT1) in HNSCC may convergently lead to the liberation of YAP1 activity. Conclusions: Collectively our data suggest that both the onset and the evolution of HNSCC might be highly dependent on (i.e., addicted to) the activation of YAP1-induced transcription. Citation Format: Muneyuki Masuda, Hirofumi Omori, Kuniaki Sato, Kenichi Taguchi, Masashige Bando, Satoshi Toh, Katsuhiko Shirahige, Koshi Mimori, Akira Suzuki. Transcriptional addiction to YAP1—a major driving force of oral cancer carcinogenesis and evolution? [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A31.

Abstract B16: Transcriptional addiction to YAP1—the major driving force of oral cancer carcinogenesis and evolution

Abstract Background: In this era of precision medicine, the clinically effective molecular target of HNSCC has not been identified so far. This is mainly because the predominant genetic alterations in HNSCC are loss-of-function (LOF) mutations in tumor suppressor genes (TSGs), which are generally undruggable targets. We hypothesized that certain effector molecules liberated from the TSGs restriction may play an essential role in HNSCC oncogenesis and evolution. In this context, we analyzed the oncogenic function of transcription coactivator YAP1, the essential downstream effector protein of the Hippo pathway. Experiments and Results: We first examined the effect of mouse tongue epithelium specific conditional double knockout (cDOK) of Mob1a/b, the core component of the Hippo pathway that inhibits YAP1, employing CreERT; Mob1aflox/flox; Mob1b−/− mouse. Consequent YAP1 activation (but not TAZ) developed extremely rapid and highly reproducible (100%) tongue tumors: carcinoma in situ (2 weeks) and invasive carcinoma (4 weeks). The tongue epithelium cells isolated from the cDOK mouse demonstrated YAP1-dependent aggressive phenotypes. In human oral HNSCC samples, expanding accumulation of nuclear YAP1 protein was observed from the basal stem/progenitor cells to invasive cancer cells and this spreading overexpression was significantly correlated with poor prognosis of patients. The YAP1 target module expression, established thorough the microarray assays with cDKO mouse cells, also correlated with the unfavorable prognosis of patients in the TCGA data. To elucidate epigenetic mechanism that underlies this extremely rapid carcinogenesis, we then conducted chip-seq for cDKO mouse cells with or without YAP1 activation. Our preliminary data indicated that YAP1-targeted genes predominantly reside in the euchromatin islands and, surprisingly, their enhancers are marked with active H3K27ac, even in the YAP1-off condition, thus ready to respond to YAP1 nuclear translocation and thereby to start transcription, contradicting our expectation that that genome-wide chromatin remodeling must be required in this rapid carcinogenesis model. Given that the presumed physiologic roles of YAP1 are a monitor of microenvironment and an emitter of regenerative cue under emergent cellular stressors (e.g., tissue injury) in the normal stem/progenitor cells, it is plausible that YAP1-target genes are kept in the ready-to-go status and constitutive activation of YAP1 and subsequent expression of targeted genes lead to the rapid carcinogenesis, causing the condition of “wounds that never heal.” Finally, our data and a recent study by Prof. Gutkind’s laboratory indicate that the major LOF of TSGs (e.g., p53 and FAT1) in HNSCC may convergently lead to the liberation of YAP1 activity. Conclusions: Collectively, our data suggest that for both the onset and the evolution, HNSCC might be highly dependent on (i.e., addicted to) the activation of YAP1-induced transcription. Citation Format: Muneyuki Masuda, Hirofumi Omori, Kuniaki Sato, Kenichi Taguchi, Masashige Bando, Satoshi Toh, Katsuhiko Shirahige, Koshi Mimori, Akira Suzuki. Transcriptional addiction to YAP1—the major driving force of oral cancer carcinogenesis and evolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B16.

Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma.

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (ANkx/ANkx) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 ANkx/ANkx) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In ANkx/ANkx mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

Abstract 1186: Regulatory role of lactate dehydrogenase A during head and neck carcinogenesis

Abstract Background: Head and Neck (H&N) cancer is one of the most prevalent neoplasms worldwide. It is well accepted that H&N cancer development is a multistep process resulting from internal and external cellular/molecular and physiological challenges leading to a number of genetic, epigenetic and metabolic abnormalities. In comparison with mitochondrial energy production in normal cells, previous studies have shown that cancer cells preferentially utilize the by-products of glycolysis, regardless of oxygen status, as an energy source. In view of this physiological variance, many investigations demonstrated that manipulations of glycolytic enzymes could potentially modulate cell malignancy of various cancers. Lactate dehydrogenase A (LDHA), one of the glycolytic enzymes essential for conversion of pyruvate into lactate, has been previously emphasized for its potential cancer-promoting effect; nevertheless, LDHA-mediated regulation for cellular malignancy and its underlying molecular mechanisms during H&N carcinogenesis is still poorly understood. Methods: Current experimental scheme was two-folds. It was initiated by histological analysis for LDHA expression in normal and tumorous tongue epithelium from animals treated with oral carcinogen 4-Nitroquinoline 1-oxide (4-NQO) to define potential role of LDHA between normal and cancerous oral tissues. Further in vitro establishment of genetic (shRNA) and chemical (oxamate) induced LDHA deficient H&N cancer cells were carried out and multifaceted cancer phenotypes including cell growth/survival, cell motility, cell differentiation and sensitivity to chemotherapeutic drug cisplatin were examined in LDHA deficient cells. Results: 4-NQO treatment stimulated tongue epithelial hyperplasia as histological analysis showed progressive dysplasia/carcinoma correlated with longer (12-week) 4-NQO treatment. LDHA expression was detected in basal layer of normal mouse tongue epithelium and enriched in suprabasal layer in 4-NQO induced hyperplastic/dysplastic tongue precancerous epithelium as well as aggressive tongue carcinoma suggesting that LDHA plays an important role during tongue carcinogenesis. In agreement with in vivo data, LDHA is highly expressed in H&N cancer cells from different origins (tongue, oropharyngeal or oral squamous cells). LDHA deficient head and neck H&N cancer cells were successfully established and showed (1) decreased cell growth; (2) downregulated cell migration, invasion and anchorage-independent growth; (3) greater expression of differentiation marker involucrin and (4) increased cisplatin sensitivity in comparison with control cells revealing LDHA-mediated tumorigenic regulations. Conclusions: Present study confirmed that suppression of glycolytic enzymes LDHA could downregulate cell malignancy in H&N cancer cells. The outcomes are of great potential to develop the metabolism mediated anti-cancer treatment for H&N cancer patients. Citation Format: Tsai-Ying Chen, Wan-Jung Chang, Chang-Yi Chen, Wan-Chun Li. Regulatory role of lactate dehydrogenase A during head and neck carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1186. doi:10.1158/1538-7445.AM2015-1186

Local hypoxia in oral mucosa (mouse) during daily fractionated irradiation – Effect of pentoxifylline

PurposeA significant reduction of radiation-induced oral mucositis by systemic application of pentoxifylline has been demonstrated in a mouse tongue model. However, the underlying mechanisms remain unclear. The present study focuses on the development of local hypoxia in mouse tongue during daily fractionated irradiation and a potential modulation by pentoxifylline. Materials and methodsDaily fractionated irradiation with 5×3Gy/week (days 0–4, 7–11) was given to the snouts of mice. Groups of 3 animals per day were sacrificed between day 0 and 14. Pentoxifylline (15mg/kg, s.c.) was administered daily from day -5 to the day before the mice were sacrificed. The expression of intrinsic hypoxia markers HIF-1α and GLUT1 in the epithelium of the lower tongue surface was analysed by immunohistochemistry in 3 animals per day; the percentage of positive epithelial cells and the staining intensity were analysed as endpoints. ResultsCompared to untreated control tissue, fractionated irradiation resulted in a progressive increase in the expression of both hypoxia markers. This effect was significantly reduced by pentoxifylline. ConclusionAn early onset of local hypoxia occurs during fractionated irradiation in mouse tongue epithelium. The effect is markedly reduced by the mucoprotective agent pentoxifylline, suggesting a mucositis-promoting role of hypoxia; this, however, deserves further investigation.

Modulation of radiation-induced oral mucositis by pentoxifylline: preclinical studies.

Oral mucositis is a frequent early side effect of radio(chemo)therapy of head-and-neck malignancies. The epithelial radiation response is accompanied by inflammatory reactions; their interaction with epithelial processes remains unclear. The aim of the present study was to investigate the effect of pentoxifylline (PTX) on the oral mucosal radiation response in the mouse tongue model. Irradiation comprised fractionation (5 fractions of 3 Gy/week) over 1 (days 0-4) or 2 weeks (days 0-4, 7-11), followed by graded local top-up doses (day 7/14), in order to generate complete dose-effect curves. PTX (15 mg/kg subcutaneously) was applied once daily over varying time intervals. Ulceration of mouse tongue epithelium, corresponding to confluent mucositis, was analyzed as the clinically relevant endpoint. With fractionated irradiation over 1 week, PTX administration significantly reduced the incidence of mucosal reactions when initiated before (day-5) the onset of fractionation; a trend was observed for start of PTX treatment on day 0. Similarly, PTX treatment combined with 2 weeks of fractionation had a significant effect on ulcer incidence in all but one experiment. This clearly illustrates the potential of PTX to ameliorate oral mucositis during daily fractionated irradiation. PTX resulted in a significant reduction of oral mucositis during fractionated irradiation, which may be attributed to stimulation of mucosal repopulation processes. The biological basis of this effect, however, needs to be clarified in further, detailed mechanistic studies.

03-P080 Reciprocal Fgf signalling interactions modulate differentiation of mouse tongue epithelium

03-P080 Reciprocal Fgf signalling interactions modulate differentiation of mouse tongue epithelium

Effects of Aging on Mouse Tongue Epithelium Focusing on Cell Proliferation Rate and Morphological Aspects

The aim of this study was to investigate cell proliferation rate and certain morphological features of mouse epithelium as aging progresses. Tongue biopsies were performed on female mice (Mus domesticus domesticus) at 2, 8, 14 and 20 months of age as indicative of adolescence, adulthood, early senescence and senescence, respectively. Histological sections of tongue were stained with hematoxylin-eosin and subjected to silver staining for active nucleolar organizer region counting. Cell proliferation rate and epithelial thickness analysis were carried out. Analysis of variance detected no differences between the groups in terms of numbers of silver-stained dots associated with nucleolar proteins. There was an increase in mean epithelial thickness in adult animals, followed by a gradual reduction until senescence. Mean keratin thickness presented an increase at 8 and 20 months of age. This difference is probably related to puberty, growth or dietary habits. Aging has no influence on oral epithelial proliferation rate in mice. A gradual reduction in epithelial thickness is a feature of aging in mammals. A conspicuous increase in the keratin layer was observed in senescence as an adaptative response to the reduction in epithelial thickness. These results suggest that aging affects the oral epithelium maturation process through a mechanism that is not related to cell proliferation.

Effects of Systemic or Topical Administration of Sodium Selenite on Early Radiation Effects in Mouse Oral Mucosa

To quantify the effect of sodium selenite (selenium) on radiation-induced oral mucositis (mouse) after subcutaneous or topical administration. Mucosal ulceration of the lower epithelium of mouse tongue was analyzed. Selenium (5 mug) was applied subcutaneously (s.c.) or locally, 60 min or 30 min prior to irradiation, respectively. In combination with single-dose irradiation, a single selenium application was given. With daily fractionated irradiation (3 Gy/fraction) for 1 week (days 0-4), selenium was administered at all 5 days of irradiation. With ten fractions over 2 weeks, selenium was applied in week 1, week 2, or both. All fractionation protocols were terminated by graded test doses to generate full dose-effect curves. In a single-dose control experiment, the ED(50) (dose after which ulcer induction is expected in 50% of the mice) was 12.9 +/- 1.6 Gy. Selenium increased the ED(50) to 17.7 +/- 2.6 Gy (s.c.; p = 0.0003) and 16.3 +/- 3.0 Gy (local; p = 0.0104). The ED(50) for test irradiation after 5 x 3 Gy was 7.4 +/- 2.2 Gy. Subcutaneous administration of selenium resulted in an ED(50) of 11.5 +/- 2.0 Gy (p = 0.0015), local application yielded an ED(50) of 10.0 +/- 2.1 Gy (p = 0.0284). The ED(50) for test irradiation after 10 x 3 Gy/2 weeks was 8.0 +/- 1.7 Gy. Subcutaneous or local administration of selenium in week 1 yielded a significant increase in ED(50) to 10.5 +/- 1.0 Gy (p = 0.0069) and 10.7 +/- 1.0 Gy (p = 0.0039), respectively. By clear contrast, selenium administration in week 2 had no significant effect. Administration in both weeks resulted in an ED(50) of 9.1 +/- 2.0 Gy (s.c.; p = 0.2747) and 9.7 +/- 1.4 Gy (local; p = 0.0541). Administration of sodium selenite during clinically relevant fractionated irradiation protocols has a significant effect during the initial treatment phase, i.e., week 1 in the mouse. Therefore, in clinical radiotherapy, the latent time to manifestation of confluent mucositis may be significantly prolonged, and hence the burden for the patient clearly reduced by selenium.

Amelioration of Early Radiation Effects in Oral Mucosa (Mouse) by Intravenous or Subcutaneous Administration of Amifostine

To quantify the reduction of radiation-induced oral mucositis by amifostine as a function of administration route. Mucosal ulceration of lower mouse tongue epithelium was analyzed. Amifostine was injected at 1.8 mg/injection subcutaneously (s.c.) or intravenously (i.v.), 45 min or 10 min prior to irradiation. With single-dose irradiation, a single amifostine injection was given. During daily fractionated irradiation (5 x 3 Gy) for 1 week, amifostine was administered s.c. or i.v. twice (days 0, 3), or s.c. on all irradiation days (days 0-4). With ten fractions over 2 weeks, five s.c. injections were given in week 1 (days 0-4) or week 2 (days 7-11), or both. Two i.v. injections were given either in week 1 (days 0, 3) or week 2 (days 7, 10). All fractionation protocols were terminated by graded test doses to generate full dose-effect curves. In a single-dose control experiment, the ED(50) (dose after which ulcer induction is expected in 50% of the mice) was 11.7 +/- 1.4 Gy. Intravenous application of amifostine increased the ED(50) to 14.0 +/- 1.4 Gy (p = 0.024), while s.c. administration had no significant effect. The ED(50) for test irradiation after 5 x 3 Gy was 5.8 +/- 1.4 Gy. Two s.c. or i.v. amifostine injections yielded ED(50) values of 7.2 +/- 1.1 Gy (p = 0.0984) or 7.6 +/- 1.2 Gy (p = 0.0334); five s.c. injections increased the ED(50) to 8.2 +/- 0.9 Gy (p = 0.0039). The ED(50) after 10 x 3 Gy/2 weeks was 6.6 +/- 1.8 Gy. Subcutaneous or intravenous administration of amifostine in week 1 yielded a significant increase in ED(50) to 9.4 +/- 2.5 Gy (p = 0.0099) and 10.0 +/- 2.2 Gy (p = 0.0014). By contrast, amifostine administration in week 2 had no significant effect. Administration in weeks 1 and 2 resulted in an ED(50) of 10.8 +/- 3.6 Gy (p = 0.0053). Amifostine during daily fractionated irradiation is effective only if administered in the initial treatment phase, i.e., week 1 in the mouse. The differences in the effect in weeks 1 and 2 suggest mechanisms of action other than radical scavenging.

Effect of EGFR-inhibition on the radiation response of oral mucosa: Experimental studies in mouse tongue epithelium

The aim was to quantify the effect of selective inhibition of the epidermal growth factor receptor (EGFR) on the radiation response of mouse oral mucosa to daily fractionated irradiation. Irradiation comprised graded single doses of 25 kV X-rays to the lower tongue surface or fractionated doses of 5 × 3 Gy week − 1 (200 kV X-rays) over 1 or 2 weeks, followed by graded local doses, to generate full dose – effect curves. For selective inhibition of EGFR, BIBX1382BS, a tyrosine kinase inhibitor, was administered orally at a dose of 50 mg kg − 1, for the entire overall treatment time. The ED50 (the dose expected to induce ulcer in 50% of the mice) for untreated mucosa was 11.9 ± 1.2 Gy. Fractionated irradiation administered over 1 or 2 weeks yielded ED50 values for the concluding test irradiation of 6.7 ± 2.1 and 6.5 ± 1.9 Gy, respectively. Administration of BIBX1382BS resulted in a non-significant increase of the top-up ED50 to 8.3 ± 1.6 Gy (p = 0.1197) after 1 week and to 7.6 ± 1.6 Gy (p = 0.2263) after 2 weeks. EGFR inhibition does not alter the radiation response of oral mucosa to fractionated irradiation or interfere with mucosal repopulation processes. This indicates that the regulation of mucosal repopulation is largely independent of EGFR activation.

Runx3 expression in mouse tongue and palate epithelium

Runx3 expression in mouse tongue and palate epithelium

Reduction of oral mucositis by palifermin (rHuKGF): Dose-effect of rHuKGF

Purpose: The aim of the present study was to determine the dose effect of palifermin (recombinant human keratinocyte growth factor, rHuKGF) for reduction of the response of oral mucosa to fractionated radiotherapy in a mouse model.Material and methods: Ulceration (confluent mucositis) of mouse tongue epithelium was analysed as the clinically relevant endpoint. Palifermin at doses from 1 – 30 mg/kg was administered before the onset (day −3), at the end of the first (day +4) or the second week of irradiation (day +11) with 5 × 3 Gy/week. Each protocol was terminated by graded radiation test (top-up) doses. In a further experiment, optimally effective doses were given on days −3 and +4, or −3, +4 and +11.Results: Single dose irradiation of mouse mucosa yielded an ED50 (dose inducing ulcer in 50% of the mice) of 10.7 ± 1.0 Gy. With fractionated irradiation for 1 week an ED50 for test irradiation (day +7) of 5.1 ± 1.9 Gy was observed. After 2 weeks (day +14), the ED50 was 7.3 ± 1.9 Gy. Palifermin significantly increased the ED50 values in all protocols tested. Maximally effective doses for single injections were 15.0 mg/kg (day −3, +11) or 22.5 mg/kg (day +4), which yielded ED50 values of 12.1 ± 1.3 Gy, 13.7 ± 1.5 Gy and 14.4 ± 1.3 Gy, respectively. Higher palifermin doses did not further increase the ED50. Repeated injections on days −3 and +4 did not increase the ED50 beyond the value obtained with injections on day +4 alone. An additional injection on day +11 increased the ED50 further to 15.1 ± 0.1 Gy.Conclusions: A significant palifermin dose-effect was seen at doses below 15 mg/kg. However, a significant increase in oral mucosal radiation tolerance by palifermin over untreated control tissue was observed already with low doses of 1 mg/kg. This indicates that in clinical studies with palifermin, the dose of the growth factor may be of minor relevance over a wide dose range.

The effect of keratinocyte growth factor on healing of manifest radiation ulcers in mouse tongue epithelium.

The purpose of this study was to examine the effect of recombinant human keratinocyte growth factor (rHuKGF) on clinically manifest acute oral mucositis. The animal model utilized in this investigation was ventral tongue epithelium of C3H/Neu mice. In a first experiment, graded single doses were applied in order to define dose effect and time course of acute mucosal ulceration, as a clinically relevant endpoint. Irradiation was given to a 3 x 3 mm2 test field in the centre of the ventral tongue with 25 kV X-rays. A single dose of 18 Gy, i.e. a dose after which ulceration is expected in more than 99% of the animals, was applied in subsequent experiments. In the study group of 20 animals, rHuKGF was applied at a daily dose of 5 mg/kg subcutaneously from the time of first diagnosis of ulcer for a maximum of 5 days. In the control group, phosphate-buffered saline was used as a placebo. The time course of ulceration, i.e. individual ulcer duration, was analysed in both the control group without rHuKGF and the study group. Irradiation with graded single doses yielded an ED50 of 11.5 +/- 0.7 Gy (logit analysis). In responding animals, the latent time to first diagnosis of ulceration and the individual ulcer duration were independent of dose. Mean latency (+/- standard deviation) was 10.5 +/- 0.5 days, mean ulcer duration 3.9 +/- 0.6 days for doses 11, 13 and 16 Gy. After a dose of 18 Gy, 39 animals developed ulceration after a mean latency of 9.3 +/- 0.3 days (control and KGF-treated). The average ulcer duration was 4.2 +/- 0.9 days in the placebo group and 4.8 +/- 0.8 days in the KGF group (P = 0.02). We conclude that when rHuKGF treatment is delayed until radiation-induced ulcers are manifest, the therapeutic activity previously reported with other treatment schedules was not observed and there was a slight prolongation of duration of ulceration. These data suggest that during tumour radiotherapy, effective rHuKGF therapy schedules should include administration before the onset of ulcerative mucositis.

Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue.

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.

A mathematical model for cell density and proliferation in squamous epithelium after single-dose irradiation

Purpose : To establish a mathematical model describing changes in cell density in squamous epithelia induced by single-dose irradiation. Detailed data from previous studies in mouse tongue epithelium have been used for this study. Materials and methods : The major mechanisms of the epithelial regeneration response, i.e. loss of division asymmetry and accelerated proliferation of stem cells, in combination with residual, abortive proliferation of sterilized cells, have been included in a tissue compartment model. These phenomena have been incorporated via three parameters: T delay, the duration of the cell cycle block; T min, the minimum stem cell cycle time due to acceleration; and T stop, the duration of abortive proliferation. The compartments introduced in the model are normal stem cells, S 1 ; sterilized stem cells, S 2 ; and post-mitotic, functional cells, F. The flux rates between the tissue compartments were defined by autoregulation of the stem cell population, and by overall cell numbers. The model was applied to fit experimental data on changes in oral mucosal cell density after single-dose exposure with 13 and 20Gy. The best-fit sets of parameters were identified by L2 norm error analysis based on the total cell count. Results : For 13 Gy, the best fit was achieved with T min = 1.0 days, T delay = 1.2 days and T stop = 7.5 days. For 20 Gy, the parameters were, T min = 0.7 days, T delay = 1.0 days and T stop = 9.5 days. In both data sets, T min was the most influential parameter. The resulting fluctuations in stem cell numbers were in good accordance with changes in radiation tolerance after 13Gy. Conclusions : The model can be used to define dose-dependent parameters describing the morphological response of squamous epithelia to single-dose irradiation. Based on these parameters, post-irradiation fluctuations in radiosensitivity can be predicted. For developing more complex and reliable mathematical models, which could incorporate transit divisions or fractionated radiotherapy, further experimental data at various dose levels are required.

温熱刺激におけるマウス舌粘膜上皮のランゲルハンス細胞とＴｈｙ‐１陽性Ｔリンパ球の免疫組織化学的研究

温熱刺激におけるマウス舌粘膜上皮のランゲルハンス細胞とＴｈｙ‐１陽性Ｔリンパ球の免疫組織化学的研究