Discussion

At the beginning of this work it was unclear how PKC gene expression was regulated. Two early studies characterised the basal promoter region of PRKCB, and showed that they had potential binding sites for a number of transcription factors [4, 161]. However, these studies did not investigate the individual contribution of these factors in regulating PKCβ gene expression, or whether their role is direct or not. Later studies showed that PKC gene expression could be regulated by mechanisms involving PKCII activity [1, 164, 262], but these studies also did not investigate the role of individual transcription factors. Therefore, in this chapter I investigated the role of Sp1. I present data clearly showing that overexpressed PKC gene expression in CLL cells is regulated by this transcription factor. This finding is consistent with the function of Sp1 because the promoter region of PRKCB

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is reported to be TATA-less [4, 161], and genes regulated by Sp1 are mostly of this type [176, 263]. Thus, I provide insight into the regulation of PKC gene expression, insight which is important to our understanding of the pathobiology of CLL cells, and also of the malignant cells of other neoplasms where PKCβII is overexpressed [149-152, 264]. Furthermore, this finding also provides insight into the effect of malignant B cells on stromal microenvironment because of a recent paper by Lutzny

et al., [144] showing that CLL cells induce the expression of PKCβII in adjacent microenvironmental stromal cells.

The results I present in this chapter strongly suggest that overexpression of Sp1 plays a major role in overexpression of PKCβII in CLL cells. My finding that Sp1 is overexpressed in CLL cells compared to normal B cells agrees with those of another study which used an RNA microarray to identify overexpressed Sp1 in CLL cells [265]. Thus, I demonstrate that Sp1 protein levels strongly correlate with PKCβII mRNA levels in CLL cells, and that Sp1 association with the PRKCB promoter drives expression of the gene. This finding is important because it suggests that overexpression of Sp1 in CLL cells may be linked to their pathophysiology. Sp1 is a pleiotropic transcription factor regulating the expression of many genes [176]. Overexpression of PKCII is a phenotypic feature that distinguishes CLL cells from other B cell malignancies [1]. The relationship between Sp1 and PKCII overexpression therefore suggests that Sp1 may be regulating the overexpression of other genes important to the phenotype of CLL cells. For example, TCL1, Lyn and Syk are all overexpressed in CLL cells, particularly in patients with progressive disease, and with disease at a late stage [266-268]. The promoter regions of these genes all have Sp1 binding sites, and, in particular, TCL1 and Syk are known to be regulated by Sp1 [269]. This suggests that regulation of PRKCB may be taken as a model to understand the phenotype of CLL cells, and possibly other tumours types, such as those associated with the lung, where increased Sp1 expression has been shown to contribute to disease progression [194].

Many of the experiments I present in this chapter use B cell lines to model the behaviour of CLL cells. MEC1 cells were derived from a patient with CLL where the malignant lymphocytes were undergoing prolymphocytic transformation [241].

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MEC1 cells retain many of the phenotypic features of CLL cells; for example, these cells are CD5 and CD19 positive, and have been used to model certain aspects of CLL cell behaviour [270-272]. Importantly, for the purposes of this Chapter, PKCII is highly expressed in this cell line. In contrast, Daudi cells were derived from a patient with Burkitt’s lymphoma and maintain a different phenotype to CLL and MEC1 cells. These cells express less PKCII than do MEC1 cells, however, they are still useful because they are easily transfected. In terms of the experiments I perform in this Chapter, these cell lines behave in a highly similar way to CLL cells. Thus, PKC gene expression is suppressed in MEC1, Daudi and CLL cells by mithramycin and Sp1-specific siRNA. Increasing concentrations of mithramycin in cultures of CLL, Daudi and MEC1 cells showed similar and proportional reductions in Sp1 and PKCβII mRNA levels in these cells. Mixed pool of siRNA nucleotides used to knockdown Sp1 expression also showed similar and proportional reductions in Sp1 and PKCβII mRNA levels in MEC1 cells, justifying the use of the mixed pool of siRNA oligos to knockdown Sp1 expression in Daudi and CLL cells. Further evidence of the similarity of MEC1 and CLL cells was gained from ChIP analysis of Sp1 association with the promoter region of PRKCB. These experiments showed that Sp1 is associated with this promoter, and can be displaced by treatment of these cells with mithramycin. Importantly, MEC1 and Daudi cells were highly useful for studying PRKCB promoter function within a luciferase assay. This type of experiment cannot be easily done using CLL cells. Taken together, these data provide a strong foundation for using MEC1 and Daudi cells to model PKC regulation in primary CLL cells. This is important for the results presented in subsequent chapters of this thesis.

The results presented in this Chapter show that Sp1 is likely to be the major driver of PRKCB transcription in CLL cells, and thereby brings insight to previous studies characterising the basal promoter region of PRKCB [4, 161]. During the preparation of the data for this Chapter, a paper by Hagiwara et al. investigating the regulation of PKC gene expression in HeLa cells was published. This paper used mithramycin, Sp1 siRNA as well as ChIP to show a role for Sp1 in regulating PKCβII expression in these cells [5]. Thus, many of the findings I present in this Chapter are confirmed by

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Hagiwara study. However, I additionally show a more direct role for Sp1 in regulating PRKCB promoter function. Mutation of the Sp1 binding sites results in complete suppression of promoter function, suggesting a dominant role for this transcription factor in regulating PKC gene expression. Similar dominant roles for Sp1 in regulating gene expression are reported for other genes in different cellular contexts [273, 274]. In particular, I demonstrate the importance of both Sp1 binding sites for PRKCB promoter function, a finding which is similar to that reported for the control of cholesterol acetyltransferase gene expression in HepG2 cells [275]. Finally, my study of PKC gene regulation is the first to demonstrate linkage between Sp1 and PKCII expression in primary cells. The study by Hagiwara et al. was performed only using a cell line.

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

Chapter Four: Investigating the potential roles of

other transcription factors in regulating the expression