Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse.

The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome.

Cytokine polymorphism frequencies in the Greek Cypriot population.

There is considerable evidence to suggest that several cytokine genes are polymorphic, resulting in differential transcription and protein expression levels among individuals. It has also been demonstrated that ethnicity can be a determinant for distinctive cytokine polymorphism frequencies. In this study, we evaluated the distribution of cytokine gene polymorphisms in 100 healthy Greek Cypriot subjects, using polymerase chain reaction-sequence-specific primers (PCR-SSP) typing analysis. Cytokine gene polymorphisms were determined for transforming growth factor (TGF) beta1 codon 10 (TGFbetac10; C to T), TGFbeta1 codon 25 (TGFbetac25; G to C), tumour necrosis factor alpha (TNFalpha) promoter -308 (G to A), interleukin (IL)-6 promoter -174 (G to C), IL-10 promoter -1082 (G to A), IL-10 promoter -819 (C to T), IL-10 promoter -592 (C to A) and interferon gamma (IFNgamma) intron 1 +874 (A to T). Frequencies for the above cytokine genotypes were calculated for the Greek Cypriot population.

Acidification and glucocorticoids independently regulate branched-chain alpha-ketoacid dehydrogenase subunit genes.

Acidification or glucocorticoids increase the maximal activity and subunit mRNA levels of branched chain alpha-ketoacid dehydrogenase (BCKAD) in various cell types. We examined whether these stimuli increase transcription of BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase plasmids containing the mouse E2 or human E1alpha-subunit promoter into LLC-PK(1) cells, which do not express glucocorticoid receptors, or LLC-PK(1)-GR101 cells, which we have engineered to constitutively express the glucocorticoid receptor gene. Dexamethasone or acidification increased luciferase activity in LLC-PK(1)-GR101 cells transfected with the E2 or E1alpha-minigenes; acidification augmented luciferase activity in LLC-PK(1) cells transfected with these minigenes but dexamethasone did not. A pH-responsive element in the E2 subunit promoter was mapped to a region >4.0 kb upstream of the transcription start site. Dexamethasone concurrently stimulated E2 subunit promoter activity and reduced the binding of nuclear factor-kappaB (NF-kappaB) to a site in the E2 promoter. Thus acidification and glucocorticoids independently enhance BCKAD subunit gene expression, and the glucocorticoid response in the E2 subunit involves interference with NF-kappaB, which may act as a transrepressor.

Regulation of expression of branched-chain alpha-keto acid dehydrogenase subunits in permanent cell lines.

The rat hepatoma cell line H4IIEC3 has demonstrated a response to both insulin and glucocorticoids in its accumulation of BCKAD subunit RNAs. It is amenable to BCKAD promoter minigene transfection analyses, demonstrating positive (glucocorticoids) and negative (insulin) regulatory effects. These cells can therefore be used as a model to identify cis-acting sites responsible for regulation of BCKAD subunit promoter activity.

Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors.

Isolation of the murine branched-chain alpha-ketoacid dehydrogenase E2 subunit promoter region.

The promoter region of the murine branched-chain alpha-ketoacid dehydrogenase E2 subunit (dihydrolipoyl transacylase) gene was isolated and characterized. Sequence analysis of the promoter-regulatory region showed the presence of two inverted 'CAAT box' sequences, the most proximal being -42 to -48 bp upstream from the determined transcription initiation site, but no TATA-box sequences, similar to the human BCKAD E2 gene. The boundary of the minimum promoter sequence appeared to reside just inclusive of this first inverted CAAT sequence, but minigene transfer analysis demonstrated that the promoter proximal between -65 and -140 bp is likely to be extremely important for controlling regulated changes in E2 RNA expression. The regulatory effect of this region may be modulated by a number of other upstream regions which were identified within the 7.0 kb sequence examined.

Effects of insulin on the regulation of branched-chain alpha-keto acid dehydrogenase E1 alpha subunit gene expression.

Alterations in dietary intake, especially of protein, may produce changes in the hepatic levels of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex. The possible role of insulin in the regulation of these observed changes in hepatic capacity for BCKAD expression was therefore examined. Steady-state RNA levels encoding three of the subunits, E1 alpha, E1 beta and E2, increased by 2-4-fold in the livers of mice starved for 3 days, a known hypoinsulinaemic state. In contrast, the levels of E1 beta and E2, but not E1 alpha, RNA were decreased when mice were fed 0% protein diets compared with the levels observed in mice fed standard (23%) or higher protein isocaloric diets. BCKAD subunit protein levels under these conditions changed co-ordinately even though the changes in RNA were not co-ordinate. The effects of hormonal changes that might be associated with these dietary changes were examined, using the rodent hepatoma cell line H4IIEC3. In these cells, the levels of E1 alpha protein and mRNA were significantly depressed in the presence of insulin. In contrast, the levels of E1 beta and E2 RNAs were not decreased by insulin. The half-lives of the E1 alpha and E2 RNAs were determined to be quite long, from 13 to 18 h, with insulin having no dramatic overall effect on the half-lives determined over 24 h. Therefore, it is likely that insulin directly affects the transcription of the E1 alpha gene rather than RNA stability in exerting its negative regulatory effect. This effect is specific to the E1 alpha subunit. The differences in BCKAD subunit RNA levels observed under various nutritional and developmental conditions may therefore be the result of the differential effects of insulin and other hormones on the multiple regulatory mechanisms modulating BCKAD subunit expression.

Molecular cloning of the murine branched chain alpha-ketoacid dehydrogenase E2 subunit: presence of 3' B1 repeat elements.

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5' sequence encoding the amino-terminal preprotein sequence and 3' untranslated region are less well conserved. The 3'-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3'-noncoding region of human E2 cDNA.

Noncoordinated responses of branched-chain alpha-ketoacid dehydrogenase subunit genes to dietary protein.

The response of the murine genes encoding the subunits of branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) to changes in dietary protein was determined. Steady-state RNA levels for two of the subunits, E1 beta and E2, decreased by two- to four-fold in the livers of mice fed 0% protein isocaloric diets compared to the levels observed in mice fed standard (23%) or high (50%) protein isocaloric diets. In contrast, the levels of RNA encoding the E1 alpha subunit did not change significantly in response to these dietary protein changes. The hepatic decreases in E1 beta and E2 RNA associated with 0% protein isocaloric diets were reversible, with prompt return to baseline levels following 48 hours of 50% protein isocaloric diets ad libitum. In kidney, no significant changes in the RNAs encoding any of the three BCKAD subunits were observed in response to changes in dietary protein. Studies of RNA variations associated with growth and development in several murine tissues, including liver and kidney, demonstrated coordinated changes between all subunits. Similar coordinated changes were observed during 3T3-L1 adipocyte differentiation. These studies suggest that the responses of the BCKAD subunit genes to alterations in dietary protein are noncoordinated and tissue-specific, in contrast to the coordinated changes observed during growth and/or differentiation. The differences in BCKAD subunit RNA levels observed under varying nutritional and developmental conditions suggest that multiple regulatory mechanisms modulate BCKAD subunit expression.

Molecular cloning and analysis of the expression of the E1 beta subunit of branched chain alpha-ketoacid dehydrogenase in mice.

The cDNA sequence encoding the murine liver E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) was determined. In the region encoding the mature E1 beta subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, rat, human, and bovine species. In contrast, they are less well conserved in the 5' sequence encoding the amino terminal preprotein sequence and 3' untranslated region. The pattern of tissue-specific expression of three BCKAD subunit RNAs was determined to be similar in both rat and murine tissues except for that observed in murine liver, where a higher than expected level of E1 beta subunit RNA was observed. Variation in the levels of this subunit might play a major role in the regulation of the overall ability of the murine liver to modulate BCKAD content in response to changing physiologic needs.