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.

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the "BAG-instructed proteasomal to autophagosomal switch and sorting" (BIPASS).

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 tetracyclineregulated 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.Cells exposed to nonlethal, elevated temperatures become transiently resistant to a subsequent heat shock. Thermotolerance development is paralleled by expression of heat shock proteins, which include members of the heat shock protein 70 (Hsp70) family (21,29). In thermotolerant cells, endogenous proteins such as p68 kinase and exogenous reporter gene products such as ␤-galactosidase and firefly luciferase show increased resistance to heat damage (4,24,27,31).It is likely that Hsp70 contributes to the protection of cellular proteins by functioning as a molecular chaperone. Such a function is implied from in vitro studies in which purified members of the Hsp70 family are found to contribute to the heat protection of several substrate proteins. Hsp70 alone can protect topoisomerase I and DNA polymerase against heat inactivation and facilitate the reactivation of heat-inactivated topoisomerase I in a concentration-dependent fashion (3, 44). For reactivation of DNA polymerase, ATP is required (44). For the recovery of heat-denatured firefly luciferase and chemically denatu...

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Molecular chaperones are involved in the protection of cells against protein damage through their ability to hold, disaggregate, and refold damaged proteins or their ability to facilitate degradation of damaged proteins. Little is known about how these processes are spatially coordinated in cells. Using a heat-sensitive nuclear model protein luciferase fused to the traceable, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now show that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. Coexpression of Hsp70, one of the major mammalian chaperones, reduced the formation of these small foci during heat shock. Instead, the heatunfolded N-luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysis demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the normal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-terminal deletion mutant of Hsp70(1-543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heatunfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleus, thereby allowing their refolding at the permissive conditions and preventing indirect damage to other nuclear components.

Irradiation of salivary glands during radiotherapy treatment of patients with head and neck cancer evokes persistent hyposalivation. This results from depletion of stem cells, which renders the gland incapable of replenishing saliva to produce acinar cells. The aim of this study was to investigate whether it is possible to expand the salivary gland stem/ progenitor cell population, thereby preventing acinar cell depletion and subsequent gland dysfunction after irradiation. To induce cell proliferation, keratinocyte growth factor (⌬N23-KGF, palifermin) was administered to C57BL/6 mice for 4 days before and/or after local irradiation of salivary glands. Salivary gland vitality was quantified by in vivo saliva flow rates, morphological measurements, and a newly developed in vitro salisphere progenitor/stem cell assay. Irradiation of salivary glands led to a pronounced reduction in the stem cells of the tissues, resulting in severe hyposalivation and a reduced number of acinar cells. ⌬N23-KGF treatment for 4 days before irradiation indeed induced salivary gland stem/progenitor cell proliferation, increasing the stem and progenitor cell pool. This did not change the relative radiation sensitivity of the stem/progenitor cells, but, as a consequence, an absolute higher number of stem/ progenitor cells and acinar cells survived after radiation. Postirradiation treatment with ⌬N23-KGF also improved gland function, and this effect was much more pronounced in ⌬N23-KGF pretreated animals. Post-treatment with ⌬N23-KGF seemed to act through accelerated expansion of the pool of progenitor/stem cells that survived the irradiation treatment. Overall, our data indicate that ⌬N23-KGF is a promising drug to enhance the number of salivary gland progenitor/stem cells and consequently prevent radiationinduced hyposalivation.

Our results show that stem cell transplantation not only rescues hypo-salivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue.

Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8⅐Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as ATP-independent molecular chaperones that can bind unfolded proteins and facilitate their processing via the help of ATP-dependent chaperones such as the Hsp70 machine, in which Bag3 may act as a molecular link between HspB, Hsp70, and the ubiquitin ligases. However, here we show that HspB8 and Bag3 act in a non-canonical manner unrelated to the classical chaperone model. Rather, HspB8 and Bag3 induce the phosphorylation of the ␣-subunit of the translation initiator factor eIF2, which in turn causes a translational shut-down and stimulates autophagy. This function of HspB8⅐Bag3 does not require Hsp70 and also targets fully folded substrates. HspB8⅐Bag3 activity was independent of the endoplasmic reticulum (ER) stress kinase PERK, demonstrating that its action is unrelated to ER stress and suggesting that it activates stress-mediated translational arrest and autophagy through a novel pathway.