retroductal-submandibular-gland-instillation-localized-fractionated

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

louisiana state university health sciences center

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.

A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells.

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.

Translation of the radioresistance kinase TLK1B is induced by gamma-irradiation through activation of mTOR and phosphorylation of 4E-BP1.

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.

Combinatorial identification of a novel consensus sequence for the covalent DNA-binding polyamide tallimustine.

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.

The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks.

Most DNA-binding ligands, ranging from protein transcription factors to small molecule antineoplastic agents, recognize duplex DNA with some degree of sequence specificity. Determining this binding specificity is important for biochemists, molecular biologists, and medicinal chemists. Some information can be obtained through the study of defined DNA sequences, but a full picture of a ligand's binding specificity can only be obtained through combinatorial means, whereby vast libraries of sequences are screened. Several combinatorial methods have been developed for the study of ligand-DNA interactions, all of which require the physical separation of ligand-bound DNA from uncomplexed DNA before amplification by PCR. Here, we describe the novel combinatorial method Restriction Endonuclease Protection Selection and Amplification (REPSA). REPSA selects for ligand-bound DNAs through their inhibition of an enzymatic process-cleavage by a type IIS restriction endonuclease-which inactivates templates for subsequent PCR amplification. We have used REPSA to identify the preferred binding sites of oligonucleotides, proteins, and small molecules on duplex DNA. Unlike conventional combinatorial methods, REPSA is amenable to the study of mixtures of native ligands with relatively unknown identities and properties. Thus, REPSA is a powerful, versatile, general method for the combinatorial determination of ligand-binding specificity and a functional means of ligand discovery.

REPSA: general combinatorial approach for identifying preferred ligand-DNA binding sequences.

The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. In this work, we show that overexpression of TLK1B hastens repair of double strand breaks (DSBs) in mouse cells. We have identified Rad9 as a protein interacting tightly with TLK1B. TLK1B phosphorylates hRad9 at S328, and the significance of this phosphorylation was addressed by expressing wild-type (WT) or mutant (S328A) hRad9 in mouse Rad9-null cells. Complementation with WT or mutant Rad9 restored the ability to survive ionizing radiation and doxorubicin, but the effect was greater for the WT protein. The S328A mutation had little effect on the level of 9-1-1 complex, but evidence shows that TLK1/1B functions to modulate the amount of 9-1-1 at DSBs. Rad9 competed with the chromatin assembly factor Asf1 for binding to TLK1B. TLK1B hastened the reassembly of nucleosomes adjacent to a DSB introduced with HO nuclease. This effect was also seen in cells expressing a kinase-dead TLK1B (KD), implicating that the kinase activity is dispensable for stimulation of chromatin remodeling at DSBs. Likewise, chromatin assembly on a plasmid was stimulated by addition of either TLK1B or KD. After the induction of DSB, occupancy of Rad9 adjacent to the break increased during repair while that of Asf1 decreased, and the effect was more pronounced in TLK1B-overexpressing cells. We propose that following genotoxic stress, TLK1/1B is first recruited to the DSB in a complex with Rad9. It then exchanges with Asf1 to promote nucleosomes eviction at the DSB and access of the repair machinery to unencumbered DNA.

Tousled homolog, TLK1, binds and phosphorylates Rad9; TLK1 acts as a molecular chaperone in DNA repair.

The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLK1/1B interacts specifically with the chromatin assembly factor Asf1, a histone H3-H4 chaperone, and with Rad9, a protein involved in DNA repair, and these interactions are believed to be responsible for the action of TLKs in double-strand break repair and radioprotection.

The expression of Tousled kinases in CaP cell lines and its relation to radiation response and DSB repair.

The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC.

Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer.

Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction.

TAT-mediated delivery of Tousled protein to salivary glands protects against radiation-induced hypofunction.

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 adenoviral-mediated 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.

Recombinant AAV9-TLK1B administration ameliorates fractionated radiation-induced xerostomia.

Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.

Article	Year
A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells. BMC cell biology\| PubMed ID: 14583098	2003
Translation of the radioresistance kinase TLK1B is induced by gamma-irradiation through activation of mTOR and phosphorylation of 4E-BP1. BMC molecular biology\| PubMed ID: 15070431	2004
Combinatorial identification of a novel consensus sequence for the covalent DNA-binding polyamide tallimustine. Biochemistry\| PubMed ID: 15709763	2005
The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks. BMC molecular biology\| PubMed ID: 16156902	2005
REPSA: general combinatorial approach for identifying preferred ligand-DNA binding sequences. Methods (San Diego, Calif.)\| PubMed ID: 17472894	2007
Tousled homolog, TLK1, binds and phosphorylates Rad9; TLK1 acts as a molecular chaperone in DNA repair. DNA repair\| PubMed ID: 18940270	2009
The expression of Tousled kinases in CaP cell lines and its relation to radiation response and DSB repair. The Prostate\| PubMed ID: 21647934	2011
Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer. The Laryngoscope\| PubMed ID: 21898433	2011
TAT-mediated delivery of Tousled protein to salivary glands protects against radiation-induced hypofunction. International journal of radiation oncology, biology, physics\| PubMed ID: 22285666	2012
Recombinant AAV9-TLK1B administration ameliorates fractionated radiation-induced xerostomia. Human gene therapy\| PubMed ID: 23614651	2013
Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells. Free radical biology & medicine\| PubMed ID: 26855419	2016

Gulshan Sunavala-Dossabhoy

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