B. Rentabilidad sobre activos
1.2 Marco conceptual 1 Administración
1.3.2 A nivel nacional
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BLOCK IN MYELOID DIFFERENTIATION OVERCOME CLINICAL REMISSIONChapter one: Introduction
1.7. CD2 expression in APL
1.7.1 Immunophenotype of APL: association of CD2 expression with the hypogranular variant subtype (M3v)
The immunophenotype of classical APL and M3v is broadly similar, being characterised by the presence of CD9, CD 13, CD33 and sialylated CD 15, with poor expression of HLA-DR (Avvisati et a l, 1992; Paietta et al, 1994; Di Noto et al, 1994; Vahdat et al,
1994). In addition, a number of studies have highlighted the expression of the T cell antigen CD2 in a subgroup of patients with APL (Claxton et a l, 1992; Biondi et a l,
1995; Guglielmi et a l, 1998). Interestingly, the initial report suggested a close relationship between the presence of a her 3 (5') PML breakpoint pattern and this phenomenon (Claxton et al, 1992). However, in this study and others it was clear that CD2 expression was not entirely restricted to patients with 5' breakpoints in PML
(Claxton et al, 1992; Maslak et al, 1993). It subsequently transpired that the correlation with PML breakpoint was most likely accounted for by a close relationship between CD2 expression and the hypogranular variant form of APL, which is typified by a high presenting WBC (Avvisati et a l, 1992) and has been associated with her 3 PML
breakpoints in some studies (Biondi et a l, 1992; Gallagher et al, 1997). The study performed by Biondi and colleagues also demonstrated that detection of CD2 in APL is a reflection of gene expression, rather than modulation of surface receptor (Biondi et al,
1995). A more recent, larger study by the same group involving 196 patients has established that CD2 expression occurs in 28% APL patients and confirmed the correlation with a her 3 PML breakpoint pattern and variant morphology (Guglielmi et a l, 1998). In particular, 58% M3v cases were found to be CD2 positive, whilst only
18% with classical morphology expressed this antigen. Furthermore, a close relationship was found between CD2 positivity and expression of the stem cell marker CD34, and the B cell antigen CD 19 (Guglielmi et al, 1998).
Therefore, whilst a number of studies have documented CD2 expression in APL, correlating it with a variety of disease characteristics, none has specifically addressed the molecular mechanisms underlying this phenomenon and its more general implications for the pathogenesis of this subtype of AML.
Chapter one: Introduction 1.7.2 Implications of CD2 expression in APL
Mechanisms by which expression of lineage-specific and lineage-restricted surface antigens are co-ordinated during haemopoietic development are incompletely understood. Characterisation of these processes is clearly critical to an understanding of leukaemogenesis. Conversely however, considerable insight into the processes regulating haemopoiesis and underlying lineage commitment have been derived from characterisation of genes disrupted by leukaemogenic translocations and subsequent mouse models using transgenic, "knock-out" and "knock-in" approaches (reviewed by Rabitts, 1994; Tenen et al, 1997; Cross & Enver, 1997). These studies, together with the characterisation of factors interacting with regulatory elements of lineage restricted genes, suggest that networks of transcription factors play a key role in mediating lineage commitment. It would appear that haemopoietic growth factors play a largely permissive role in this process; although this remains an area of some contention (reviewed by Metcalf, 1998; Enver et a l, 1998). Distinct experimental approaches have shown that a variety of genes whose expression is considered to be lineage restricted are co-expressed in multipotential haemopoietic progenitor cells (Ford et al, 1992; Jimenez et al, 1992; Cross et a l, 1994; Ford et a l, 1996). In particular, studies employing DNase I hypersensitivity assays established that the regulatory regions of p-globin, CD3S, IgH and myeloperoxidase (MPO) genes lie within open chromatin in multipotential murine FDCP cells (Ford et al, 1992; Jimenez et a l, 1992; Ford et al, 1996). These cells have the advantage that they can be induced to differentiate along various lineages with exposure to appropriate haemopoietic growth factors, thereby permitting investigation of the mechanisms underlying lineage commitment and consolidation of a given differentiation pathway. Interestingly, the IgH regulatory regions were found to become DNase I insensitive following induction of myeloid differentiation, whilst the CD35 enhancer remained sensitive under these circumstances, only becoming insensitive following progression along the B lineage pathway (Ford et a l, 1992). Further investigation of the mechanisms underlying expression of MPO, has revealed that despite its accessibility to chromatin, the enhancer is functionally inactive in multipotential progenitors, becoming active only following commitment to the myeloid lineage (Ford et a l, 1996). Pu.l and C-EBP binding sites were identified in the enhancer, prompting the suggestion that regulation of MPO expression could be mediated through exchange of C-EBP isoforms at enhancer elements, which could reflect temporally regulated variations in expression level, cellular localisation and phosphorylation status, thereby influencing binding characteristics; and could also depend upon the phosphorylation status of Pu.l (Ford et al, 1996). It may be envisaged
Chapter one: Introduction that similar mechanisms may underly the regulation of other lineage restricted genes associated with commitment to a specific differentiation pathway. More recent studies have further extended the findings of DNase I hypersensitivity assays, which were performed in populations of multipotential murine haemopoietic progenitors, by employment of an RT-PCR approach to analyse expression patterns of arrays of lineage associated genes in single multipotential progenitor cells (Cheng et a l, 1996; Hu et al,
1997). Whilst it remained a possibility that the results derived from DNase I hypersensitivity assays could have been explained by the presence of subpopulations of cells already committed to specific lineages, the single cell RT-PCR approach was able to confirm co-expression of p-globin, myeloperoxidase and various lineage associated cytokine receptors in multilineage progenitors, through analysis of both murine cell lines and CD34+/lin- selected marrow cells (Hu et al, 1997). Similarly, single-cell RT-PCR analysis of human CD34+/CD38- selected progenitors demonstrated co-expression of a number of lineage-restricted transcription factors, including Pu.l, GATA-2 and SCL (Cheng et al, 1996). These findings prompted a model whereby lineage commitment is determined by achievement of a threshold level of co-operating key transcription regulators which, re-enforced by positive and negative feedback loops, generate "programs" inducing and leading to maintenance of expression of the respective lineage associated genes, whilst mediating to varying degree repression of genes associated with other lineages (Cross & Enver, 1997; Enver & Greaves, 1998).
With more widespread application of immunophenotyping techniques to facilitate leukaemia diagnosis, it became apparent that antigens previously considered lymphoid- specific could be detected in cases of AML. For many years, it has been debated as to whether such "biphenotypic" leukaemias represent either expansions of rare stem cells that co-express lymphoid and myeloid antigens ("lineage promiscuity" model(Greaves et al, 1986)), or arise in lineage-committed cells in which the presence of "inappropriate" surface markers reflect aberrant gene expression associated with leukaemogenesis ("lineage infidelity" model (Smith et a l, 1983)). In this context the presence of CD2 on some APL blasts is particularly intriguing since this subtype of AML is relatively well differentiated. Indeed, whilst studies performed in NOD/SCID mice have suggested that in the majority of cases of AML, the leukaemogenic event occurs at the level of primitive haemopoietic progenitors (CD344-/CD38-); blasts derived from APL patients failed to engraft in the mice suggesting that in this form of AML committed progenitors are targeted (Bonnet & Dick, 1997). Consistent with this hypothesis, CD34+/CD38- progenitors derived from patients with APL were found to be negative for the PML-
Chapter one: Introduction
RARa fusion gene, which was restricted to the committed CD34+/CD38+ marrow
population (Turhan e ta l, 1995).
1.7,3 Regulation of CD2 expression
In order to begin to address the mechanism underlying CD2 expression in APL it is important to determine how the gene is regulated during normal haemopoietic development. The structure of the CD2 gene has been defined, comprising 5 exons spanning a genomic region of 15 kb (Diamond et a l, 1988; Lang et a l, 1988). DNase I hypersensitivity assays have established the presence of two distinct hypersensitive sites denoted DHS 1 & 2 situated 5' to the gene and a further series of sites lying 3' to the coding sequence, denoted DHS 3 (see Figure 1.6) (Wotton et a l, 1989; Festenstein et al, 1996). DNase I hypersensitive sites are known to occur around regions of chromatin that are not tightly packed into nucleosomes, identifying the position of regulatory elements (Elgin, 1988; Gross & Garrard, 1988). A 28.5kb genomic fragment encompassing theCD2 coding sequence and DHS 1, 2 and 3 was found to permit position independent, copy number dependent, tissue specific (i.e. restricted to T cell lineage) expression in transgenic mice, thereby implying that all the key regulatory elements required for appropriate expression of the gene are contained within this region (Lang et a l, 1988; Greaves et a l, 1989). The 3' hypersensitive sites were found to correspond to enhancer elements and a locus control region (LCR), conferring high-level tissue specific expression of a CD2 transgene, independent of the site of integration (Greaves et a l, 1989; Lake et al, 1990). A subsequent study using high resolution DNase I hypersensitivity assays has established that there are actually 3 clusters of hypersensitive sites within the 3' flanking region, denoted HS 1-3 (Festenstein et a l,
1996). It is now clear that the LCR function is localised within the HS 3 region; which is essential to establish an open chromatin configuration and possesses no intrinsic enhancer activity. This would suggest that the LCR functions in the establishment &/or maintenance of an open chromatin domain acting in conjunction with adjacent tissue specific enhancers contained within HSl of the DHS 3 region which are essential for mediating high level gene expression through an interaction with promoter elements (Festenstein e ta l, 1996; Kioussis & Festenstein, 1997).