Rescue of Salivary Gland Function after Stem Cell Transplantation in Irradiated Glands

Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome). In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit+ cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency

Isomorphism between Systems of Equivariant Singularities

AbstractIn this article isomorphisms between systems of singularities equivariant under different Lie group actions are investigated and a sufficient condition for two systems to be isomorphic is given. With this sufficiency theorem we show that the system ofO(n)-equivariant singularities in its irreducible representation on Rnis isomorphic to that of one-dimensional Z2-equivariant singularities and the system of[formula]-dimensionalO(n)-equivariant singularities is isomorphic to that ofn-dimensionalSn-equivariant singularities

Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G2/M checkpoint control in Drosophila cells

Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall ( stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 ( two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.</p

SENSING AND REROUTING OF PROTEIN DEGRADATION TOWARDS AUTOPHAGY UPON PROTEASOMAL IMPAIRMENT

The accumulation of misfolded, mutant proteins is a common basis for many adult onset neurodegenerative diseases. Cells have evolved an elaborate protein quality control system, which acts to facilitate the folding or refolding of misfolded protein species by molecular chaperones or, if folding is unsuccessful, these same chaperones often target the misfolded proteins for degradation, thereby preventing protein aggregation. Intracellular degradation is primarily mediated by two proteolytic systems: the autophagy and the ubiquitin proteasomal systems. Proteotoxic stress can lead to proteasomal impairment and augmented authophagosomal capacity in order to ensure proper clearance of clients (proteasome-autophagy switch). However, neither the mechanism of sensing nor that of switching is understood. Here, we show that the ER is main sensor for proteasomal inhibition through the IRE-1alpha-Xbp-1 signalling cascade. After proteasome inhibition, BAG-3 is upregulated in a HSF-1 independent manner, but in a Xbp-1 dependent manner and is a major executor of the proteasome-autophagy switch. BAG-3 both boosts autophagy and redirects HSP70-bound proteasomal clients to autophagosomes through competitive inhibition with its family member BAG-1, that normally directs HSP70-bound clients to the proteasome, thus playing a key role in the maintenance of protein homeostasis under proteotoxic stress conditions

In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells

Thermal Protein Denaturation and Protein Aggregation in Cells Made Thermotolerant by Various Chemicals: Role of Heat Shock Proteins

Thermotolerance (TT) induced by sodium arsenite (A-TT: 100 mu M, 1 h, 37 degrees C), ethanol (E-TT: 6% (v/v), 25 min, 37 degrees C), and diamide (D-TT: 300 mu M, 1 h, 37 degrees C) was compared to heat-induced thermotolerance (H-TT: 15 min, 44 degrees C) using HeLa S3 cells. All four pretreatments led to comparable levels of thermotolerance and also induced resistance to arsenite-, ethanol-, and diamide-induced toxicity (clonogenic ability). Stress-induced expression of the major heat shock proteins (hsp27, hsc70((p73)), hsp70((p72)), and hsp90) was generally highest in H-TT cells and lowest in A-TT cells. Interestingly, the four types of TT cells showed distinct differences in certain aspects of resistance against thermal protein damage. Thermal protein denaturation and aggregation determined in isolated cellular membrane fractions was found to be attenuated when they were isolated from H-TT and A-TT cells but not when isolated from E-TT and D-TT cells. The heat resistance in the proteins of the membrane fraction corresponded with elevated levels of hsp70(p72) associated with the isolated membrane fractions. In the nuclear fraction, only marginal (not significant) attenuation of the formation of protein aggregates (as determined by TX-100 (in)solubility) was observed. However, the postheat recovery from heat-induced protein aggregation in the nucleus was faster in H-TT, E-TT, and D-TT cells, but not in A-TT cells. Despite the fact that elevated levels of hsp27, hsp70((p73)), and hsp70((p72)) were found in the TX-100 insoluble nuclear fraction derived from all TT cells, no correlation was found with the degree of resistance in terms of the accelerated recovery from nuclear protein aggregation. The only correlation between accelerated recovery from nuclear protein aggregates was that with total cellular levels of hsp27. The data indicate that heat-induced loss of clonogenic ability may be a multitarget rather than a single target event. A threshold of damage may exist in cells after exposure to heat; multiple sets of proteins in (different compartments of) the cell need to be damaged before this threshold is exceeded and the cell dies. As a consequence, stabilization of only one of these sets of proteins is already sufficient to render cells thermotolerant at the clonogenic level. (C) 1995 Academic Press, Inc