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Background: In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-Like Kinase (TLK1B) and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line.

Oral complications of salivary hypofunction often afflict cancer patients undergoing radiotherapy for head and neck cancers. Dry mouth or xerostomia is an undesirable consequence of radiotherapy that compromises normal oral functions in addition to causing odynophagia and increasing the patient's risk of oral infections and dental caries. Radiation-induced xerostomia is irreversible, and palliative measures to provide symptomatic relief remain the mainstay of treatment. Previously, we identified a splice variant of a cellular kinase, Tousled-like kinase 1B (TLK1B), which when overexpressed protects normal epithelial cells against ionizing radiation (IR)-induced cell death. To address the need to protect salivary glands in patients undergoing regional radiotherapy, we investigated whether preemptive expression of TLK1B in salivary glands protects against IR. In stably-derived salivary cell lines in vitro, TLK1B expression increased cell survival after IR. Cells expressing exogenous TLK1B were less radiosensitive (A5-TLK1B, a/b¼0.67 Gy; ParC5-TLK1B, a/b¼4.3 Gy) compared to control cells (A5-BK, a/b¼1.7 Gy; ParC5-BK, a/b¼32.7 Gy). Using a recombinant adenovirus serotype 5 viral vector for TLK1B gene transfer into rat submandibular salivary glands in vivo, we demonstrated that TLK1B protects the saliva-secreting acinar cells and better preserves salivary gland function against IR relative to control glands. After a single fraction of 16 Gy, the decline in salivary function at 8 weeks was less pronounced in TLK1B-treated animals (40%) as compared to saline-treated controls (67%). Histopathological analysis demonstrated increase in acinar atrophy, decrease in acinar cell number, and increase in inflammatory infiltrate and fibrosis in irradiated control tissues relative to TLK1B-treated glands. These results show the radioprotective benefits of TLK1B and implicate its usefulness in the management of regional radiotherapy-induced xerostomia.

Background: The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes probably, due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases it's resistance to ionizing radiation (IR) or the radiomimetic drug doxorubicin, also likely due to changes in chromatin remodeling. TLK1B is translationally regulated by the availability of the translation factor eIF4E, and its synthesis is activated by IR. The reason for this mechanism of regulation is likely to provide a rapid means of promoting repair of DSBs. TLK1B specifically phosphorylates histone H3 and Asf1, likely resulting in changes in chromatin structure, particularly at double strand breaks (DSB) sites.

Background: The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and chromosome missegregation probably due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect.

Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.

Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviralmediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (10 11 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline-and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p < 0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection against radiation assists in preserving submandibular gland function. AAV2/9-TLK1B treatment could prove beneficial in attenuating xerostomia in patients with head and neck cancer undergoing radiotherapy.

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