Cancer is of paramount medical concern as an increasingly major contributor of disease-related fatalities of significant prevalence - particularly in the context of current statistical/stochastical epidemiological studies which predict that one in three people will contract cancer at some stage of their lives, whilst one in four of these patients will die as a consequence of their particular neoplastic-associated condition.
The human Rad9 protein exists in two full-length isoforms (termed Rad9A and Rad9B) whose respective differentially-elevated levels and related expression profiles are distinctive for specific tumour cell tissue types.
Most known functions of the DNA damage response protein Rad9 are executed via the well-characterised Rad9-Radl-Husl ("9-1-1") protein complex, which is loaded onto chromatin in close vicinity to DNA lesion sites.
The chromatin-loaded "9-1-1" complex functions as both a DNA damage "sliding-clamp" sensor and a recruitment platform which modulates and co-ordinates the activities of a wide variety of different proteins implicated in cell cycle checkpoint signalling, steroidal nuclear receptor signalling, protein chaperoning and DNA repair - via associative protein-protein interactions with
the C-terminal tail domain of the Rad9 sub-unit.
This toroidal, heterotrimeric "9-1-1" DNA sliding-clamp complex is highly conserved and its recently resolved crystal structure shows a functional similarity to the homotrimeric PCNA DNA sliding-clamp complex.
Associative ring formation amongst the individual Rad9, Rad1 and Hus1 sub-units is limited, via stringent steric and thermodynamic parameters, to the heterotrimeric type "9-1-1" DNA slidingclamp complex configuration.
Recent clinical data strongly indicate that over-expression of Rad9, but not Hus1 or Rad1, also promotes tumour growth.
Aside from the well-documented phenomena of the modulation of apoptotic signalling and pyrimidine nucleobase biosynthesis activities, comparatively little is known about the "9-1-1" complex-independent functions of the human Rad9 protein - whose dysfunctional activities may be implicated in the development and progression of carcinogenesis.
The initial research emphasis of this Ph.D. project was focused on the elucidation on the mechanism of expression and potential functional roles of a novel N-Terminal truncated ("short") variant of the Rad9 protein - termed "Rad9-.S.", which is expressed in the experimental eukaryotic cell cycle model organism Schizosaccharomyces pombe.
Expression of relatively low levels of a constitutive form of Rad9-S were detected in S. pombe cells under normal conditions, whilst significantly increased levels of an expressed inducible form of the protein were detected in S. pombe cells as part of an exclusive response to heat shock.
In addition to Rad9-S, the constitutive expression of two shorter truncated Rad9 variants - termed Rad9-VS and Rad9-I ("very-short" and "tiny"), was also detected in S. pombe cells.
This experimental observation indicates that S. pombe may prove to be a useful homologous eukaryotic model organism, in future research studies, for the elucidation of the unknown functions of the four truncated isoform variants of human Rad9B which may be implicated in novel mechanisms of carcinogenic development and progression.
The mechanism of expression of the Rad9-S, Rad9-VS truncated protein variants was postulated to involve alternative translation at the alternative AUG start-codon sites at Methionine 50 (M50) and Methionine 74 (M74) within the S. pombe rad9 gene, in which leaky ribosomal scanning is implicated.
Heat shock may increase the frequency of leaky ribosome scanning, exclusive to the M50 alternative AUG start-codon site, via alterations of the secondary topological configuration of the rad9 mRNA, in which rad9 mRNA-protein associative interactions with heat-shock proteins, RNA chaperones and/or RNA stabilisers may also be implicated.
The rad9 mRNA region spanning the codon region Ml to M50 inclusive may also function as a novel type of cis-acting hypothermic suppressor element that induces a supramolecular configurational rearrangement of the mRNA secondary structure in response to cold shock which blocks leaky ribosome scanning at the alternative M50 AUG start-codon site with consequential suppression ofRad9-S expression under low (l6°C) and moderate temperatures (25-30°C).
Expression of the Rad9-T truncated protein variant was postulated to involve a limited proteolytic cleavage mechanism in which metacaspase-mediated and/or COP9 signalosome-mediated limited proteolytic processing may be implicated.
In silico sequence alignment analyses identified a potential metacaspase target-site motif within the S. pombe Rad9 protein and comparative modelling indicated that key residues at the focal cleavage-site of this motif were situated at the Rad9:Radl interface of the "9-1-1" DNA sliding-clamp complex.
In silico multiple sequence alignment analyses also indicated that the S. pombe Rad9 protein contained two sequences, flanking the identified potential metacaspase target-site motif, which exhibited significant homology with the two alternative C-termini of the H. sapiens Rad9B paralogue and its truncated isoforms.
Distinctive phosphorylation-type post-translational modifications of Rad9-S were also found to influence the expression of the respective two smaller truncated protein variants; Rad9-VS and Rad9-T.
It was postulated that a variety of potential Rad9 phospho-isoforms may be implicated in a interactive "activity-modulatory feedback" mechanism, in which they function as transcriptional and/or translational regulators of the expression of the Full-length Rad9, NL\49-Rad9 ("Rad9-S") and NL\ 73-Rad9 ("Rad-VS").
Whilst kinase-mediated phosphorylation of several key residues, identified within a conserved potential metacaspase site in the full-length Rad9, Rad9-S and Rad9-VS protein isoforms, may inhibit proteolytic-cleavage formation of the detected Rad9-T truncated protein variant.
Taken together, these data indicated that specific checkpoint kinase-mediated phosphorylation of the S. pombe Rad9 protein may constitute part of a "feedback" signalling network for the regulation of metacaspase-mediated processing of the S. pombe Rad9 protein which may be implicated in novel checkpoint responses that suppress the formation and/or alter specific
functional activities of the Rad9-Rad1-Hus1 ("9-1-1") DNA sliding-clamp and promote the proteolytic expression of two truncated Rad9 isoforms whose functions are unknown, but may elicit alternative regulatory cell cycle signalling pathways under particular genotoxic and/or environmental stress conditions.
Whilst confirmation of the existance of these two postulated metacaspase-generated truncated Rad9 isoforms and elucidation of their roles in cell cycle checkpoint signalling remains to be established, their potential novel functions may be analogous to those of the H. Sapiens Rad9B isoforms which are as yet unknown.
S. pombe cells "Cre-Lox"- engineered for the exclusive expression of the Rad9-S protein variant exhibited a high degree of cytotoxic sensitivity towards a wide range of different types of genotoxic agents.
This supported the initial hypothesis which postulated that deletion of the first 49 N-terminal amino acid residues in the truncated Rad9-S protein variant would sterically suppress the formation of the constrained ring configuration of the Rad9-Radl-Husl heterotrimeric DNA sliding-clamp complex and thus render the engineered cells unable to elicit the appropriate cell cycle checkpoint responses to various types of induced DNA damage.
An unanticipated, exceptional experimental observation was the partial resistance of engineered Rad9-S cells (~30% retained cell viability) towards acute exposure to the Topoisomerase I (Topo I) inhibitor drug camptothecin (~30% retained cell viability) - which transiently traps the enzyme on the DNA in S-phase.
Consequential collision of DNA replication forks with the resultant duplex-immobilised DNACPT- Topo I ternary complex leads to the formation of one-sided double-stranded breaks which are subsequently detected by the G2-M DNA damage checkpoint
Five key phosphorylation sites were identified within the truncated Rad9-S protein variant which were critical for the partial resistance of engineered Rad9-S cells towards camptothecin-induced DNA damage - notably; Y12 and Y62 (potential Mph1 kinase target sites) and Tl 76, T363, S374 (equivalent Rad3 kinase target sites to those found in the full-length Rad9 protein at positions
T225, T412 and S423 respectively).
A potential DNA-binding domain, spanning residues M50 - M74 Rad9, was also identified within the truncated Rad9-S protein that may possess additional functions as a nuclear translocation signal responsive element, a nuclease recruitment-site and/or exonuclease catalytically-active site which are implicated in the co-ordinated repair of camptothecin-induced DNA double-stranded breaks.
Subsequent genetic and biochemical experimental data indicated that the truncated Rad9-S protein variant may mediate a co-ordinated cell cycle arrest signal and DNA repair response to camptothecin-induced DNA damage, that functions outside of the canonical Rad9-Rad1-Hus1 complex, which is channelled into a novel pathway that interacts independently of both the G2-M
and mitotic checkpoints.
Comparative acute survival assays performed on cultures of the S. pombe cells engineered for the exclusive expression of the truncated Rad9-S protein variant, under induced osmotic stress conditions, rendered the cells hyper-sensitive to camptothecin-induced DNA damage and hyper-resistant to heat shock.
Comparative acute survival assays also indicated that deletion of the Sty 1 kinase, but not Wis1 kinase, within an exclusive Rad9-S expression type genetic background also increased the sensitivity of the cells to camptothecin-induced DNA damage.
Whilst comparative bioinformatics-based in silico sequence alignments of the Rad9-S, Sty1 and Wis1 indicated that both Rad9-S and Sty1 contained potential interactive motifs for the Rad3 kinase, which were absent in the Wis1 protein.
Taken together, these data indicated that the truncated Rad9-S protein variant may be implicated in two novel separate differential signalling responses upon exposure to either heat shock or camptothecin, in which appropriate pathway selection is dictated via the suppression or induction of Rad9:Sty1-mediated co-operative activation of the Rad3 kinase.
Comparative acute survival assays indicated that deletion of rad1 within an exclusive Rad9-S expression type genetic background rendered the engineered cells hypersensitive to heat shock, but had no adverse impact on their observed partial resistance to camptothecin-induced DNA damage.
Comparative acute survival assays also indicated that deletion of hus1 within an exclusive Rad9-S expression type genetic background rendered the engineered cells hypersensitive to both hyperthermic- and camptothecin- induced genotoxic cytological stresses.
Experimental genetic studies also indicated that the Rad17 clamp-loader protein is unlikely to be directly implicated in these Rad9-S-mediated checkpoint responses to hyperthermic- or camptothecin- induced genotoxic stresses.
Taken together, these experimental observations indicate that the Rad9-S- mediated response to camptothecin-induced DNA damage proceeds via formation of a heterodimeric, "open-ring", Rad9-S:Hus1 C-clamp type complex, whilst the Rad9-S- mediated response to hyperthermic stress proceeds via formation of an alternative heterotrimeric Rad9-S:Rad1 :Hus1 toiroidal clamp
complex and that these respective checkpoint pathways are novel functions of Rad9 which operate outside of the canonical full-length Rad9-Rad1-Hus1 heterotrimeric DNA sliding-clamp complex.
It was also postulated that the shorter truncated isoform variants Rad9-VS and Rad9-T may be implicated in a feedback regulatory mechanism for modulatory control of the respective functional activities of the Rad9-S protein-mediated responses to both beat shock- and camptothecin- induced types of DNA damage - which may also be may also be analogous to those of the human Rad9B paralogue and its respective truncated isoforms.
Future experimental studies into the identified S. pombe Rad9-S- modelled checkpoint response to camptothecin-induced genotoxicity may offer potential new insights into the reasons for the exclusive elevated expression of Rad9, but neither Rad1 or Hus1, in aggressive breast tumours and/or novel mechanisms of acquired tumour drug resistance to camptothecin-based anti-cancer
chemotherapeutics.
Whilst future experimental investigation of the identified S. pombe Rad9-S- modelled checkpoint response to hyperthermic stress may advance knowledge of the complex mechanisms of action implicated in the combinatorial hyperthermic-enhanced efficacy of chemo- and/or radiotherapeutic adjuvant clinical anti-cancer regimens and provide useful information of the acquired
mechanisms of tumour resistance to these types of treatments.
Taken together, in summarised context, progressive research into the initial "pilot data" acquired from this Ph.D. project may provide vital information for the future treatment of chronic breast cancer patients administered camptothecin-derivatised agents, in combination with adjuvant hyperthermotherapy and/or radiotherapy, to combat the metastatic spread of refractory tumours which have developed multiple drug resistance to the "conventional arsenal" of chemotherapeutic drugs utilised in standard clinical practice - such as taxols and anthracyclics.