Medicine & Health

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Now showing 1 - 8 of 8
  • (2009) Gan, Pei Pei
    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

  • (2008) Schuller, Christine
    In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

  • (2009) Liaw, Tracy Yun En
    Tubulin-binding agents (TBAs) are employed widely in cancer treatment, including childhood acute lymphoblastic leukaemia (ALL). Despite their success, resistance to TBAs can be a major clinical problem. Mechanisms mediating resistance to TBAs that target the colchicine-binding site on β-tubulin, along with novel therapeutic strategies were investigated. 2-Methoxyestradiol (2ME2) is a multi-targeted TBA active in various cancer types, yet its efficacy and mechanisms(s) of action in haematological malignancies are not well evaluated. To improve understanding of mechanisms(s) of action and drug resistance, leukaemia cells, CCRF-CEM, were selected for resistance to 2ME2. These resistant cells displayed reduced drug-induced mitotic arrest and increased tubulin polymer levels. Moreover, the resistant cells also acquired β-tubulin mutations that affected microtubule stability and induced conformational change(s) to the 2ME2-binding site on β-tubulin. Two of the mutations resided in the colchicine-binding site, yet did not affect colchicine sensitivity, suggesting that 2ME2 and colchicine differ in their binding to β-tubulin. These 2ME2-resistant leukaemia cells have provided novel insights into microtubule stability and drug-target interactions to the tubulin molecule. Differential proteomics analysis identified alterations in the expression of cytoskeletal proteins in the 2ME2-resistant leukaemia cells, including a significant increase in class II β-tubulin. Furthermore, γ-enolase and galectin-1 were up-regulated and class IVb β-tubulin was down-regulated in 2ME2-resistant cells. Gene specific knockdown of class II β-tubulin expression in neuroblastoma cells using small interfering RNA increased sensitivity to 2ME2 and other colchicine-binding agents, suggesting that class II β-tubulin plays a functional role in drug resistance and sensitivity. In a clinically relevant NOD/SCID ALL mouse xenograft model, orally active 2ME2 and its analogue, ENMD-1198, increased survival rates in human ALL xenografts compared to control mice. ENMD-1198 showed better activity than 2ME2 against the ALL xenografts. Combining two microtubule-destabilising agents, ENMD-1198 and vincristine, demonstrated synergistic effect by causing microtubule disruption, angiogenesis and cell proliferation inhibition, and apoptosis induction in leukaemia cells. Importantly, ENMD-1198 and vincristine combination was more potent than single agent treatment in a vincristine-resistant ALL xenograft. In conclusion, this thesis identified novel mechanisms of resistance to 2ME2 and developed a new therapeutic strategy that could improve the treatment of drug refractory childhood ALL.

  • (2006) Po'uha, Sila Tu'ipolotu
    Antimicrotubule drugs are used in the treatment of childhood neuroblastoma and acute lymphoblastic leukaemia (ALL). Resistance to these agents can be a major clinical problem and mechanisms mediating resistance are not fully understood. Previous studies have reported an association between the actin cytoskeleton and resistance to antimicrotubule drugs. Thus, the aim of this study was to investigate the role of the actin regulating proteins, LIM kinases (LIMK1 and LIMK2) in drug resistance. In addition, the role of γ actin, a major actin isoform, in drug resistance was also examined. Chapter 1 reviewed the known mechanisms of antimicrotubule drug resistance and the interaction between the microtubules and actin cytoskeleton. The methodologies used in this study are described in chapter 2. LIMKs are known to regulate the actin cytoskeleton via phosphorylation of cofilin. Real Time RT PCR and western blotting was used in chapter 3 and showed that expression of LIMKs and their downstream target cofilin was altered in antimicrotubule resistant neuroblastoma and leukaemia cells. Moreover, altered LIMK expression was detected in in vivo derived vincristine resistant ALL xenografts and ALL clinical samples, further demonstrating that alterations in LIMKs and cofilin are associated with antimicrotubule drug resistance. Importantly, in chapter 4, gene silencing and drug treated clonogenic assays were performed to elucidate the functional role of LIMK1 and LIMK2 in drug response. Silencing of LIMK1 and/or LIMK2 increased sensitivity of neuroblastoma cells to microtubule targeting drugs and DNA damaging agents, suggesting that LIMKs may be useful targets to improve the efficacy of anticancer drugs. γ-Actin has been associated with drug resistance and chapter 5 used gene silencing and drug treated clonogenic assays to show that decreased γ actin expression conferred resistance to anitmicrotubule drugs but not to DNA damaging agents. Microscopy and tubulin polymerisation assays showed that reduced γ-actin protects microtubules from paclitaxel induced polymerisation. This data supports a functional role for γ actin in antimicrotubule drug action. In conclusion, this study showed that LIMKs and γ actin mediate the action of antimicrotubule drugs and other anticancer agents, demonstrating that the actin cytoskeleton may serve as a useful drug target to improve the efficacy of anticancer drugs.

  • (2009) Markovic, Ana
    Acute lymphoblastic leukaemia (ALL) is the most common cause from death of disease in children. Whilst cure rates over the last 30 years have drastically improved, the children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments. The role that vascular endothelial growth factor (VEGF), an integral component of both neovascularisation and normal haematopoiesis, plays in the progression and invasiveness of solid tumours is well established. However, its function in haematological malignancies has been a more recent and thus less considered observation. Human leukaemia cells secrete VEGF, which may act in a paracrine manner with the bone marrow microenvironment to promote the survival and proliferation of leukaemia cells. In addition to VEGF being produced by leukaemias, it also increases vascularity in the bone marrow and lymph nodes of patients. Our previous work has established a panel of 10 childhood acute lymphoblastic leukaemia xenografts from patient biopsies in NOD/SCID mice. Several of these secrete VEGF, and express the FMS-like tyrosine kinase-3 (FLT-3). FLT 3, a receptor tyrosine kinase (RTK), and its ligand, play an essential role in regulating normal haematopoiesis. This thesis builds on the previous work by examining the relationship between VEGF and FLT 3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients. The results show that the high expression and activation of FLT 3, significantly increases the secretion VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of a class III receptor tyrosine kinase (RTK) inhibitor (SU11657), humanised anti-FLT 3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT 3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous gene expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FL on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells. Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional chemotherapy are warranted.

  • (2009) Tee, Eu Leong Andrew
    This thesis showed that the expression of tissue transglutaminase (TG2) was commonly reactivated by HDACIs and repressed by Myc oncoproteins in neuroblastoma and breast cancer cells. The repression of TG2 expression was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. N-Myc acted as a transrepressor by recruiting the HDAC-1 protein to the TG2 gene core promoter. In neuroblastoma-bearing N-Myc transgenic mice, HDACI treatment induced TG2 expression and demonstrated marked anti-tumour activity in vivo. Intriguingly, recent literature suggests that the TG2 precursor mRNA is alternatively spliced to generate a full-length (TG2-L) and a short form (TG2-S) mRNAs. Distinct from TG2-S protein, the TG2-L protein possesses a GTP-binding site at Arg580, which inhibits transamidation activity when GTP bound. The current study demonstrated that when TG2-L is repressed, or, TG2-S or the GTP-binding site-mutant (TG2-L-R580A) is over-expressed, there is spontaneous neuroblastoma cell differentiation. In addition, the investigations in this thesis showed that cell differentiation induced by TG2-S and the TG2-L-R580A is dependent on TG2 transamidation activity, and that TG2-L blocked neuroblastoma cell differentiation by repressing gene expression of vasoactive intestinal peptide (VIP). Repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation induced by N-Myc siRNA and retinoid differentiation therapy. Moreover, combination therapy with the HDACI, Trichostatin A (TSA), and interferon-α (IFNα) co-operatively inhibited the expression of TG2 and pro-angiogenic genes in neuroblastoma cells in vitro, and inhibited tumour angiogenesis in neuroblastoma-bearing N-Myc transgenic mice. In conclusion, N-Myc blocks neuroblastoma cell differentiation by repressing TG2 gene expression via the recruitment of the HDAC-1 protein to the TG2 gene core promoter. The data suggest that Myc oncoproteins may possess a more widespread capacity for transcriptional suppression by recruiting HDAC-1 proteins to target gene promoters. The opposing effects of TG2-L and TG2-S on neuroblastoma cell differentiation are due to a structural difference in the GTP-binding Arg580 residue. TG2-L and TG2-S represent novel drug development targets for the treatment of Myc-induced malignancies and enhancing retinoid differentiation therapy. Combination therapy with HDACIs and IFNα could be an effective therapeutic strategy for the treatment of neuroblastoma.

  • (2008) Wilson, Carmen Louise
    In Australia, up to 80% of individuals diagnosed with childhood cancer are now expected to survive for more than five years after their initial diagnosis. However, survivors of childhood cancer are at risk of developing late sequelae as a consequence of therapies received during childhood. The aim of this study was to determine the incidence of selected late sequelae in a cohort of Australian childhood cancer survivors and identify treatment and genetic factors that may modify the risk of late sequelae in survivors. Our study included 1150 individuals treated for childhood cancer at the Sydney Children’s Hospital between 1962 and 1999, who had remained in remission >3 years and were confirmed to be alive. Rates of mortality and second cancers among survivors were compared against population rates to determine standardised mortality and incidence ratios. Survivors completed a questionnaire on the incidence of adverse health conditions and provided a buccal specimen. Real time PCR was used to detect polymorphisms in genes involved in drug detoxification and transport. Rates of mortality and secondary cancers were found to be 7.5-fold (95%CI 5.4-10.1) and 4.9-fold (95%CI 2.9-8.0) higher among survivors of childhood cancer relative to the general population, respectively, with the highest risks observed for those survivors previously treated for Hodgkin’s disease. Over 60% of survivors reported at least one cardiopulmonary, endocrine or sensory-motor condition following diagnosis of childhood cancer; the most frequently observed conditions included growth hormone deficiency, hypothyroidism, and hypertension. Late sequelae were most frequently reported by females and survivors of brain tumours. Genetic investigations showed that an increased risk of growth hormone deficiency was associated with homozygosity for the GSTM1 null polymorphism, while no gene associations were observed to influence the risk of second cancers among survivors. Our study demonstrates that survivors of childhood cancer are at risk of developing a variety of health conditions as a result of anti-cancer therapies received during childhood. Determining risk factors for late sequelae based on therapy type, lifestyle and genetic predisposition will enable the optimisation of treatment protocols and promote the future well-being of childhood cancer survivors.

  • (2009) Davies, Carwyn
    The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.