Kathleen W. Scotto

Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals

The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.

Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y.

ABSTRACT Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level ofMDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

Transcriptional Activation of the MDR1 Gene by UV Irradiation ROLE OF NF-Y AND Sp1

The MDR1 promoter is subject to control by various internal and external stimuli. We have previously shown that the CCAAT box-binding protein, NF-Y, mediates MDR1 activation by the histone deacetylase inhibitors, trichostatin A and sodium butyrate, through the recruitment of the co-activator, P/CAF. We have now extended our investigation to the activation of MDR1 by genotoxic stress. We show that activation of the MDR1 promoter by UV irradiation is also dependent on the CCAAT box (−82 to −73) as well as on a proximal GC element (−56 to −42). Gel shift and supershift analyses with nuclear extracts prepared from human KB-3-1 cells identified NF-Y as the transcription factor interacting with the CCAAT box, while Sp1 was the predominant factor binding to the GC element. Mutations that abrogated binding of either of these factors reduced or abolished activation by ultraviolet irradiation; moreover, co-expression of a dominant-negative NF-Y protein (NF-YA29) reduced UV-activated transcription. Interestingly, YB-1, a transcription factor that also recognizes the CCAAT motif and had been reported to mediate induction of the MDR1 promoter by ultraviolet light, was incapable of interacting with the double-stranded MDR1 CCAAT box oligonucleotide in nuclear extracts, although it did interact with a single-stranded oligonucleotide. Furthermore, a mutation that abolished activation of MDR1 by UV-irradiation had no effect on YB-1 binding and co-transfection of a YB-1 expression plasmid had arepressive effect on UV-inducible transcription. Taken together, these results indicate a role for both NF-Y and Sp1 in the transcriptional activation of the MDR1 gene by genotoxic stress, and indicate that YB-1, if involved, is not sufficient to mediate this activation.

Association of Sorcin With the Cardiac Ryanodine Receptor

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A >500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

Histone Methyltransferase MLL1 Regulates MDR1 Transcription and Chemoresistance

The multidrug resistance 1 gene (MDR1) encodes P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) transporter family that confers tumor drug resistance by actively effluxing a number of antitumor agents. We had previously shown that MDR1 transcription is regulated by epigenetic events such as histone acetylation, and had identified the histone acetylase P/CAF and the transcription factor NF-Y as the factors mediating the enzymatic and DNA-anchoring functions, respectively, at the MDR1 promoter. It has also been shown that MDR1 activation is accompanied by increased methylation on lysine 4 of histone H3 (H3K4). In this study, we further investigated histone methylation in MDR1 regulation and function. We show that the mixed lineage leukemia 1 (MLL1) protein, a histone methyltransferase specific for H3K4, is required for MDR1 promoter methylation, as knockdown of MLL1 resulted in a decrease in MDR1 expression. The regulation of MDR1 by MLL1 has functional consequences in that downregulation of MLL1 led to increased retention of the Pgp-specific substrate DIOC(2)(3), as well as increased cellular sensitivity to several Pgp substrates. Regulation of MDR1 by MLL1 was dependent on the CCAAT box within the proximal MDR1 promoter, similar to what we had shown for MDR1 promoter acetylation, and also requires NF-Y. Finally, overexpression of the most prevalent MLL fusion protein, MLL-AF4, led to increased MDR1 expression. This is the first identification of a histone methyltransferase and its leukemogenic rearrangement that regulates expression of an ABC drug transporter, suggesting a new target for circumvention of tumor multidrug resistance.

Identification of Function for CD44 Intracytoplasmic Domain (CD44-ICD) MODULATION OF MATRIX METALLOPROTEINASE 9 (MMP-9) TRANSCRIPTION VIA NOVEL PROMOTER RESPONSE ELEMENT

Background: CD44, a multifunctional receptor, undergoes cleavage to produce an intracytoplasmic domain (CD44-ICD) that translocates into the nucleus. Results: CD44-ICD binds to a novel DNA consensus sequence and activates many genes. Conclusion: We finally explain the multifunctionality of CD44 and reveal new genes affected by CD44. Significance: Our findings will accelerate the understanding of how CD44-ICD regulates a multitude of cell functions. CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.

Caffeine Regulates Alternative Splicing in a Subset of Cancer-Associated Genes: a Role for SC35

ABSTRACT Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity, playing a key role in development, gene expression and, when aberrant, human disease onset. Many of the factors involved in alternative splicing have been identified, but little is known about their regulation. Here we report that caffeine regulates alternative splicing of a subset of cancer-associated genes, including the tumor suppressor KLF6. This regulation is at the level of splice site selection, occurs rapidly and reversibly, and is concentration dependent. We have recapitulated caffeine-induced alternative splicing of KLF6 using a cell-based minigene assay and identified a “caffeine response element” within the KLF6 intronic sequence. Significantly, a chimeric minigene splicing assay demonstrated that this caffeine response element is functional in a heterologous context; similar elements exist within close proximity to caffeine-regulated exons of other genes in the subset. Furthermore, the SR splicing factor, SC35, was shown to be required for induction of the alternatively spliced KLF6 transcript. Importantly, SC35 is markedly induced by caffeine, and overexpression of SC35 is sufficient to mimic the effect of caffeine on KLF6 alternative splicing. Taken together, our data implicate SC35 as a key player in caffeine-mediated splicing regulation. This novel effect of caffeine provides a valuable tool for dissecting the regulation of alternative splicing of a large gene subset and may have implications with respect to splice variants associated with disease states.

Differential regulation of MDR1 transcription by the p53 family members: Role of the DNA binding domain

Although the p53 family members share a similar structure and function, it has become clear that they differ with respect to their role in development and tumor progression. Because of the high degree of homology in their DNA binding domains (DBDs), it is not surprising that both p63 and p73 activate the majority of p53 target genes. However, recent studies have revealed some differences in a subset of the target genes affected, and the mechanism underlying this diversity has only recently come under investigation. Our laboratory has demonstrated previously that p53 represses transcription of the P-glycoprotein-encoding MDR1 gene via direct DNA binding through a novel p53 DNA-binding site (the HT site). By transient transfection analyses, we now show that p63 and p73 activate rather than repress MDR1 transcription, and they do so through an upstream promoter element (the alternative p63/p73 element (APE)) independent of the HT site. This activation is dependent on an intact DNA binding domain, because mutations within the p63DBD or p73DBD are sufficient to prevent APE-mediated activation. However, neither p63 nor p73 directly interact with the APE, suggesting an indirect mechanism of activation through this site. Most interestingly, when the p53DBD is replaced by the p63DBD, p53 is converted from a repressor working through the HT site to an activator working through the APE. Taken together, these data indicate that, despite considerable homology, the DBD of the p53 family members have unique properties and can differentially regulate gene targeting and transcriptional output by both DNA binding-dependent and -independent mechanisms.

Transcriptional regulation of MDR genes

The emergence of resistance in a tumor population is most often associated with a disregulation of gene expression, usually at the level of transcription. A major goal in the field of cancer chemotherapy is to define the mechanisms underlying transcriptional regulation of drug resistance genes in an effort to identify targets for therapeutic intervention. Recently, considerable progress has been made in identifying the molecular mechanisms involved in the transcriptional regulation of the P-glycoprotein (Pgp) gene. When overexpressed in tumor cells, Pgp confers resistance to a variety of chemotherapeutic agents; this resistance has been termed MDR (multidrug resistance). Moreover, Pgp is a normal component of a variety of highly differentiated cell types and, as such, is regulated by both internal and external environmental stimuli. In this review, we will discuss the current knowledge regarding the DNA elements and protein factors involved in both constitutive and inducible regulation of Pgp transcription in normal and tumor cells.

The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines.

BACKGROUND/PURPOSE Neuroblastoma is the most solid common extracranial malignancy in childhood. Despite multimodality treatment, high-risk disease continues to carry a poor prognosis. Glucocorticoids have been shown previously to induce differentiation in murine neuroblastoma cell lines, but no such effect has been documented in human neuroblastoma cells. Glucocorticoids are known to be active in the differentiation process of the neural crest. These studies describe the effects of dexamethasone on 6 human neuroblastoma cell lines. METHODS Dexamethasone was added to cultured neuroblastoma cell lines (LA1-5S, LA1-15N, BE[2]S, BE[2]N, SH-EP-1, SH-SY5Y) maintained in media supplemented with either normal serum or charcoal-depleted serum. Proliferation assays were performed, and flow cytometry was used to assess alterations in cell cycle. Cells were closely monitored for morphological changes with serial phase-contrast microscopy. Immunohistochemistry (3F8, NF-1, TRK-A) of cultured cells was used to evaluate differentiation. Glucocorticoid receptor levels was assessed using immunoblotting. RESULTS Dexamethasone decreased the rate of cellular proliferation in both standard and charcoal-depleted conditions. Flow cytometry showed a G1 accumulation. Increased expression of the differentiation-associated antigens was found in cells cultured in charcoal-depleted media, and a further augmentation was seen with the addition of dexamethasone. In standard media, dexamethasone had no detectable effect on the expression of these antigens. Glucocorticoid receptor expression was found to be comparable in all cell lines. CONCLUSIONS Human neuroblastoma cells are sensitive to the differentiating effects of dexamethasone in an environment of charcoal-depleted serum. This phenomenon may be caused by the existence of growth and mitogenic factors in serum that are inhibiting differentiation.