Cancer is currently the leading cause of death by disease in children under the age of 15 in the US. While the number of childhood cancer survivors continues to grow, psychological research on this population has lagged. Existing research on the psychosocial effects of childhood cancer is marked by inconsistent conclusions as well as methodological limitations. However, the effect of childhood cancer on social functioning is one area with relatively more consistency. Existing research suggests that childhood cancer can lead to deficits in prosocial skills as well as the emergence of social problems. The present study investigated individual change in social functioning for five children diagnosed with Acute Lymphoblastic Leukemia ALL) over the first year of treatment compared to healthy control peers. Children with cancer demonstrated a decrease in social activity as well as an unexpected increase in social skills not demonstrated by healthy control children.

Diss. (sammanfattning) Uppsala : Univ., 2009. / Härtill 4 uppsatser.

T-ALL accounts for 15% of childhood leukemias and approximately 60% of patients overexpress TAL1. TAL1/SCL encodes a transcription factor that regulates hematopoiesis by dimerizing with additional transcription factors including E12, E47, and GATA-1. TAL1 has also been found to repress expression of NF-κB1, potentially promoting formation of an NF-κB p65/c-Rel heterodimer that encourages cell survival by up-regulating IAPs and IκB. However, the correlation between TAL1 and p65/c-Rel expression and their effects on downstream targets like IKK, IκB, and other anti-apoptotic proteins is poorly understood. Jurkat cells, expressing TAL1, were treated with TNFα and/or etoposide to induce apoptosis and experiments were performed to assess the expression of proteins of interest. Caspase-8 activity assays were also performed to help delineate the apoptotic signal present in these cells. Determining if interactions between TAL1, NF-κB, and other downstream targets help promote apoptotic resistance will further research into better, more targeted treatments for T-ALL. / Department of Biology

T-cell acute lymphoblastic leukemia (T-ALL) is a childhood cancer created through genetic alterations; most commonly upregulation of TALI and LMOI oncoproteins. T-ALL is treated with radiation and chemotherapy, but malignant T-cells are resistant to apoptotic stimulation. To study this disorder, AKR-DP-603 cells were transduced to express both oncoproteins. Western blots verified protein expression and each population was treated with etoposide. Caspase-3 and Annexin-V/FITC apoptosis assays were performed following treatment. When the response of control cells was compared to engineered cells, no difference was observed from the Annexin-V/FITC assay, and only LM01 cells showed a difference in the caspase-3 assay. Furthermore, cells were transfected with siRNA to TALI and LM01 and the apoptotic response was re-tested. Complete silencing was verified by Western and apoptotic activity varied in the TALI population for both assays. These differences might indicate that cells resisted etoposide induction and following silencing were sensitized apoptotic induction. / Department of Biology

Pediatric acute lymphoblastic leukemia (ALL) is the most common malignancy in children, which results from the malignant transformation of progenitor cells in the bone marrow into leukemic cells. The precise mechanisms for this transformation are not well defined, however recent studies suggest that aberrant regulation of gene expression or DNA methylation may play an important role. Hence, the aim of this thesis was to use novel methods to investigate genome-wide gene expression and DNA methylation patterns in a large collection of primary ALL cells from pediatric patients. With these studies, we aimed to increase the understanding of factors that regulate gene expression and DNA methylation in ALL. In the first study of the thesis we found that data obtained from genome-wide digital gene expression analysis enabled excellent cytogenetic subtype-specific classification of ALL cells and revealed new features of gene expression within the disease, such as prevalent antisense transcription and alternative polyadenylation. In the second study we used technology developed for large-scale single nucleotide polymorphism (SNP) genotyping for quantitative analysis of allele-specific gene expression (ASE), revealing widespread ASE in ALL cells. Analysis of DNA methylation in promoter regions of the genes displaying ASE using DNA-microarrays revealed frequent regulation of gene expression by DNA methylation. In the third study, using the same DNA methylation array, we identified differences in the DNA methylation patterns in ALL cells at diagnosis compared to healthy mononuclear cells from the bone marrow of the same children at remission. In the fourth study we measured the DNA methylation of >450,000 CpG sites across the genome in a large collection of ALL samples and non-leukemic control cells. We found that ALL cells displayed highly divergent DNA methylation patterns depending on their cytogenetic subtype and widespread regions of differential methylation were enriched for repressive histone marks. DNA methylation levels at distinct regions in the genome were substantially increased at relapse compared to matched cells from diagnosis. Collectively, the results presented in this thesis provide new insights into the patterns of gene expression and epigenetic changes in ALL and further increase our understanding of the development and progression of the disease, which will hopefully lead to better treatment options in the future.

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.

Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.

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.

[Truncated abstract] Acute lymphoblastic leukaemia (ALL) is the most common form of cancer that affects children and the leading cause of child cancer-related death. There have been dramatic improvements in the 5-year event free survival (EFS) for childhood ALL in recent years, with EFS reaching 75-90% for some forms of the disease. Despite this success, treatment for the disease is aggressive with numerous long and short-term side effects. Many cases of ALL are characterised by chromosomal defects including translocations, variations in chromosome number and the deletion of the tumour suppressor genes. Although these gross chromosomal changes have been extensively studied in childhood ALL, the cascade of altered gene expression that results from these changes has not. Further improvements in survival and the quality of life of survivors relies on a better understanding of the underlying biology of ALL. The primary aim of this study was to determine the gene expression profile of pre-B ALL specimens and normal, or non-malignant, control cells using microarrays in order to further examine the underlying biology of childhood ALL. ... Analysis of the ALL profile with two normal haematopoietic populations demonstrated that ALL specimens have a profile similar to that of CD34+ cells. Specifically, specimens of the MLL subtype had a profile that uniformly resembled that of CD34+ cells. Other subgroups contained specimens with profiles that ranged in similarity to that of CD34+ cells, however, the gene expression profile of all ALL specimens analysed more closely resembled the CD34+ cells than the more differentiated CD19+IgM- cells. This study identified exceptionally high expression of connective tissue growth factor (CTGF/CCN2) in ALL specimens compared to control cells. CTGF expression was v restricted to B-lineage ALL specimens, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies by Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. The study presented here provides insight into the biology of ALL including the observation that ALL cells have an immature gene expression profile similar to that of CD34+ cells and the possible existence of an autocrine loop involving CTGF. The findings may also have clinical application in the future treatment of ALL, such as the use of metabolic inhibitors or the blocking of CTGF expression. This study provides an important insight into many aspects of ALL disease biology and may offer potential new therapeutic targets for the treatment of ALL.

Thesis (Ph. D.)--University of North Texas, Aug., 2007. / Title from title page display. Includes bibliographical references.