Cindy S. Malone

Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells

Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.

Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma

The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated JH gene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.

CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific B29 (Igβ/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells. B29 expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced B29 promoters: at conventional CpG and at CC(A/T)GG B29 promoter sites. The pattern of methylated CpG (mCpG) and CmC(A/T)GG B29 promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of CmC(A/T)GG sites in the B29 promoter significantly repressed in vivo transcriptional activity. Also, methylation of a central conserved CmCTGG B29 promoter site blocked the binding of early B cell factor. This methylated motif formed DNA–protein complexes with nuclear extracts from all cell types examined. Therefore, CmC(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA–protein complex formation.

An Upstream Oct-1- and Oct-2-Binding Silencer Governs B29 (Igβ) Gene Expression

The B cell-specific B29 (Igβ) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5′ of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

Genes expressed during the IFNγ-induced maturation of pre-B cells

Interferon-gamma (IFN gamma) exerts diverse responses in B cell development ranging from growth arrest and apoptosis to proliferation and differentiation. IFN gamma stimulates murine 70Z/3 pre-B cells to express surface immunoglobulin (Ig) and this system serves as a useful model for the pre-B to immature B cell transition in B cell development. To analyze this developmental transition, we used a PCR-based subtractive hybridization in combination with miniarray screening to identify differentially-expressed genes in IFN gamma-stimulated compared with unstimulated 70Z/3 pre-B cells. The majority (44%) of the differentially-expressed genes obtained were known IFN gamma-inducible. These included multiple isolates from each of three multi-gene families, including two guanylate-binding protein (47 and 67kDa GBP) families of GTPases and the hematopoietic IFN gamma-inducible nuclear protein family (HIN-200). These multiple isolates of genes comprised the majority of the total isolated and sequenced clones. Other known IFN gamma-induced genes in this group included Ig kappa light chain and Ly-6, as well as genes with functions in antigen processing, cellular regulation, and cytoskeletal organization. Another 36% of the genes identified were previously known, but not known to be IFN gamma-inducible (e.g. pre-B cell enhancing factor, PBEF). The remaining 20% of the IFN gamma-induced isolates did not match entries in Genbank, and thus, may represent novel genes involved in IFN gamma responses and/or in the pre-B to immature B cell transition. Overall, the majority of the individual genes isolated were either not known to be IFN gamma responsive or were not previously known.

Defining the Role of Oxygen Tension in Human Neural Progenitor Fate

Summary Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2α-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined, these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here, we show that low O2 tension and hypoxia-inducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies, we implicate O2 tension as a modifier of a key cell fate decision, namely whether neural progenitors differentiate toward neurons or glia. Furthermore, our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage. We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types.

Bob1 (OCA-B/OBF-1) Differential Transactivation of the B Cell-Specific B29 (Igβ) and mb-1 (Igα) Promoters

The B29 (Igβ) and mb-1 (Igα) gene products are B cell-specific essential components of the B cell receptor that are coexpressed at all stages of B cell differentiation, with the exception of plasma cells, which lack mb-1 expression. Transcription of both genes is governed by a similar cassette of interactive transcription factor-binding elements, including octamer motifs, in TATA-less promoters. In this study, we show the B cell-specific B29 gene promoter is transactivated in B and non-B cells by cotransfection with the B cell-specific octamer cofactor gene, Bob1 (OCA-B/OBF-1). The expression of Bob1 is also sufficient to override the silencing effects of the B29 silencer. This indicates that Bob1 plays a critical role in B cell-specific B29 promoter expression. In contrast, coexpression of Bob1 had no effect on mb-1 promoter activity. Bob1 transactivation only occurs with select octamer sequences that have an adenosine at position 5 (ATGCAAAT). The B29 promoter conforms to this consensus octamer motif, while the mb-1 promoter octamer motif does not. Octamer motif swapping between B29 and mb-1 promoters renders B29 unresponsive to Bob1 transactivation and makes mb-1 competent for Bob1 transactivation, thereby indicating that the B29 octamer motif is solely responsible for Bob1 interaction. Additionally, the mb-1 construct containing the B29 octamer motif is expressed in a plasmacytoma cell line, while the wild-type mb-1 promoter is not. Bob1 transactivation of B29 and the lack of this transactivation of mb-1 account for the differential expression of B29 and mb-1 in terminally differentiated plasma cells.

Epigenetic Silencing of Stk39 in B-Cell Lymphoma Inhibits Apoptosis from Genotoxic Stress

B-cell lymphomas, the most frequent human immune system malignancies, often contain dysregulated TCL1 oncogene expression. TCL1 transgenic (TCL1-tg) mice develop a spectrum of B-cell malignancies, supporting an oncogenic role for TCL1 in B cells. Our prior global survey of DNA methylation patterns in TCL1-tg B-cell lymphomas identified many lymphoma-specific candidate hypermethylated genes, including Stk39. The Stk39 encoded protein, sterile 20-like-related proline-alanine-rich kinase (SPAK), regulates cell stress responses, and microarray studies identified reduced SPAK expression in metastatic prostate and treatment-resistant breast cancers, suggesting that its loss may have a role in cancer progression. Here we identified DNA hypermethylation and SPAK silencing in TCL1-tg B-cell lymphomas and SPAK silencing without DNA methylation in multiple subtypes of human B-cell lymphomas. SPAK knockdown by shRNA protected B cells from caspase-dependent apoptosis induced by DNA double-strand breaks but not apoptosis in response to osmotic or oxidative cell stressors. Caspase 3 activation by cleavage was impaired with SPAK repression in DNA damaged B cells. Interestingly, c-Jun NH(2)-terminal kinase is potentially activated by SPAK and pharmacological inhibition of c-Jun NH(2)-terminal kinase in SPAK-expressing B cells recapitulated the cell-protective phenotype of SPAK knockdown. Taken together, these data indicate that SPAK loss in B-cell lymphomas promotes increased cell survival with DNA damage and provides a potential mechanism for increased resistance to genotoxic stress in cancer.

Gene expression patterns in AIDS versus non-AIDS-related diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is more prevalent and more often fatal in AIDS patients compared to immune-competent individuals. Potential explanations for these differences include distinct tumorigenic mechanisms and/or altered cellular microenvironments. We previously discovered that the TCL1 (T-cell leukemia-1) proto-oncogene is expressed in a high proportion of AIDS-DLBCL compared to DLBCL cases and that aberrant TCL1 expression causes DLBCL in a new transgenic mouse model. Here, we continue to search for other genes that may contribute to the differential pathogenesis of DLBCL in AIDS. Gene subtraction yielded over 1800 potential AIDS-DLBCL candidates, of which about 50% were unknown and not further considered. The remaining 50% of genes were annotated and, when combined with miniarray screening from multiple patient samples, were reduced to 18 candidate genes for extended analysis. These 18 genes showed distinct patterns of expression in both AIDS-DLBCL and DLBCL samples. However, unlike TCL1, none of these genes was preferentially associated with either AIDS-DLBCL or DLBCL. Our data suggest that the increased incidence and severity of AIDS-DLBCL compared to DLBCL is likely due to crippled immune surveillance rather than to markedly different gene expression profiles.

An essential octamer motif in the mb-1 (Igα) promoter

Abstract The mb-1 (Igα) gene is B cell-specific and expressed throughout B cell maturation. In combination with B29 (Igβ) and surface immunoglobulin mb-1 comprises the B cell receptor complex (BCR). The murine mb-1 promoter has been characterized to depend on the trans-acting transcription factors; Sp1, ets, lkaros, and EBF for full promoter activity. These trans-acting factors are also involved in the regulation of mb-1’s closely related heterodimeric partner, B29. However, octamer transcription factors 1 and 2 (Oct-1 and Oct-2) are also necessary for full B29 promoter activity while they are not known to be required for mb-1 promoter activity. Here, we show that the octamer transcription factors bind a degenerate octamer consensus sequence within the mb-1 promoter. Like B29, the mb-1 octamer-binding, motif interacted with both ubiquitously expressed Oct-1 and the B cell-specific Oct-2 transcription factors. Furthermore, the interaction of Oct-1 and Oct-2 contributed to the regulation of the mb-1 promoter as site-directed mutations within the octamer motif substantially reduced its activity. These data confirm that octamer factor interactions and function contribute to the full transcriptional activity of the mb-1 promoter.