Betty H. Johnson

Biological activities of oxysterols

Literature dealing with the biological activities of cholesterol autoxidation products and related oxysterols in vivo and in vitro published since the previous 1981 monograph is reviewed. Although several oxysterols are important cholesterol metabolites implicated in bile acids and steroid hormones biosynthesis, effects on cellular membranes and on specific enzyme systems as well as cytotoxic, atherogenic, mutagenic, and carcinogenic activities characterize oxysterols as a class. Circumstantial evidence implicates oxysterols of the human diet and those formed in vivo with human health disorders, but recent work also supports an hypothesis that some oxysterols be endogenous intracellular regulators of de novo sterol biosynthesis. The true physiological relevance, if any, of these matters has not been adduced.

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.

Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity

Noncoding RNAs have drawn significant attention in biology recently. Whereas the current research is highly inclined to microRNAs, research on other noncoding RNAs has lagged behind. Here, we investigated a novel noncoding RNA that has been known as precursor microRNA miR-886 (pre-miR-886). Pre-miR-886 has been proposed also as a vault RNA, a component of the vault complex implicated in cancer drug resistance. We identified pre-miR-886 as a 102-nucleotide-long, abundant cytoplasmic RNA that is neither a genuine pre-microRNA nor a vault RNA. Pre-miR-886 is physically associated with PKR (Protein Kinase RNA-activated), an interferon-inducible and double-stranded RNA dependent kinase. The suppression of pre-miR-886 activates PKR and its downstream pathways, eIF2α phosphorylation and the NF-κB pathway, leading to impaired cell proliferation. We also found that pre-miR-886 is suppressed in a wide-range of cancer cell lines and in clinical specimens. This study is the first intense characterization of pre-miR-886 as well as the initial report on its function as a PKR regulator, which suggests a critical role in tumorigenesis.

Pathway interactions between MAPKs, mTOR, PKA, and the glucocorticoid receptor in lymphoid cells

BackgroundGlucocorticoids are frequently used as a primary chemotherapeutic agent in many types of human lymphoid malignancies because they induce apoptosis through activation of the glucocorticoid receptor, with subsequent alteration of a complex network of cellular mechanisms. Despite clinical usage for over fifty years, the complete mechanism responsible for glucocorticoid-related apoptosis or resistance remains elusive. The mitogen-activated protein kinase pathway is a signal transduction network that influences a variety of cellular responses through phosphorylation of specific target substrates, including the glucocorticoid receptor. In this study we have evaluated the pharmaceutical scenarios which converge on the mitogen-activated protein kinase pathway to alter glucocorticoid sensitivity in clones of human acute lymphoblastic CEM cells sensitive and refractory to apoptosis in response to the synthetic glucocorticoid dexamethasone.ResultsThe glucocorticoid-resistant clone CEM-C1-15 displays a combination of high constitutive JNK activity and dexamethasone-induced ERK activity with a weak induction of p38 upon glucocorticoid treatment. The cells become sensitive to glucocorticoid-evoked apoptosis after: (1) inhibition of JNK and ERK activity, (2) stimulation of the cAMP/PKA pathway with forskolin, or (3) inhibition of mTOR with rapamycin. Treatments 1–3 in combination with dexamethasone alter the intracellular balance of phospho-MAPKs by lowering JNK phosphorylation and increasing the level of glucocorticoid receptor phosphorylated at serine 211, a modification known to enhance receptor activity.ConclusionOur data support the hypothesis that mitogen-activated protein kinases influence the ability of certain malignant lymphoid cells to undergo apoptosis when treated with glucocorticoid. Activated/phosphorylated JNK and ERK appear to counteract corticoid-dependent apoptosis. Inhibiting these MAPKs restores corticoid sensitivity to a resistant clone of CEM cells. Forskolin, which activates the cAMP pathway, and rapamycin, which inhibits mTOR, also inhibit JNK. Further, the sensitizing treatments result in a largely dexamethasone-dependent increase in the total pool of glucocorticoid receptor phosphorylated at serine 211. The phospho-serine 211 receptor is known to be more potent in activating gene transcription and apoptosis. The interactive effects demonstrated here in reverting resistant cells to corticoid sensitivity could provide therapeutic clinical potential in the treatment of lymphoid malignancies.

Oxysterol-induced cell death in human leukemic T-cells correlates with oxysterol binding protein occupancy and is independent of glucocorticoid-induced apoptosis

In eukaryotic cells oxysterols inhibit cholesterol biosynthesis and cell growth. A potent oxysterol, 25-hydroxycholesterol, was used to investigate the biological effects of oxysterols on three clonal lines of either glucocorticoid-sensitive or -resistant CEM cells, human leukemic T-lymphocytes. In addition, the glucocorticoid sensitivity of an oxysterol-resistant CEM cell line was tested. Oxysterols blocked growth and caused the lysis of cells regardless of their glucocorticoid response. All cells studied herein possessed an oxysterol binding protein with high affinity for 25-hydroxycholesterol. For all clones grown in serum-free medium, the half-maximal cytolytic concentration of 25-hydroxycholesterol (20-40 nM) correlated with its affinity (Kd = approximately 31 nM) for this oxysterol binding protein. Both cholesterol and mevalonate reversed 25-hydroxycholesterol cytotoxicity; 3-6 microM cholesterol or 0.1 mM mevalonate decreased 60 nM 25-hydroxycholesterol cytotoxicity by 50%. This cholesterol or mevalonate reversal appeared possible even after several days of 60 nM oxysterol treatment. The protective effect of cholesterol could be overcome by increasing 25-hydroxycholesterol concentrations. Cholesterol and mevalonate did not prevent glucocorticoid-mediated lymphocytolysis. Furthermore, the oxysterol-resistant line was sensitive to dexamethasone lysis. These data support the hypothesis that oxysterols and glucocorticoids act independently to block the growth of human leukemic lymphoblasts.

Protein Kinase A, not Epac, Suppresses Hedgehog Activity and Regulates Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia Cells

Cyclic AMP synergizes strongly with glucocorticoids (GC) to induce apoptosis in normal or malignant lymphoid cells. We examined the individual roles that cAMP-dependent protein kinase (PKA) and Epac (exchange protein directly activated by cAMP), two intracellular cAMP receptors, play in this synergistic effect. Our studies demonstrate that PKA is responsible for the observed synergism with GC, whereas Epac exerts a weak antagonistic effect against GC-induced apoptosis. We find that endogenous PKA activity is higher in the GC-sensitive clone than in the GC-resistant clone. In the GC-sensitive clone, higher PKA activity is associated with lower Hedgehog (Hh) activity. Moreover, inhibition of Hh activity by Hh pathway-specific inhibitors leads to cell cycle arrest and apoptosis in CEM (human acute lymphoblastic leukemia, T lineage) cells, and the GC-sensitive clone is more sensitive to Hh inhibition. These results suggest that Hh activity is critical for leukemia cell growth and survival and that the level of Hh activity is in part responsible for the synergism between cAMP and GC.

Gene networks in glucocorticoid-evoked apoptosis of leukemic cells.

To discover the genes responsible for the apoptosis evoked by glucocorticoids in leukemic lymphoid cells, we have begun gene array analysis on microchips. Three clones of CEM cells were compared: C7-14, C1-15 and C1-6. C7-14 and C1-15 are subclones from the original clones C7 (sensitive to apoptosis by glucocorticoids) and C1 (resistant). C1-6 is a spontaneous revertant to sensitivity from the C1 clone. Previously we presented data on the sets of genes whose expression is altered in these cell clones after 20 h exposure to dexamethasone (Dex). The two sensitive clones, which respond by undergoing apoptosis starting about 24h after Dex is added, both showed >2.5-fold induction of 39 genes and 2-fold reduction of expressed levels from 21 genes. C1-15, the resistant clone, showed alterations in a separate set of genes. In this paper, we present further analysis of the data on genes regulated in these cell clones after 20 h Dex and compare them with the genes regulated after 12h Dex. Some, but not all the genes found altered at 20 h are altered at 12h, consistent with our hypothesis that sequential gene regulation eventually provokes full apoptosis. We also compare the levels of basal gene expression in the three clones. At the basal level no single gene stands out, but small sets of genes differ >2-fold in basal expression between the two sensitive and the resistant clone. A number of the genes basally higher in the resistant clone are potentially anti-apoptotic. This is consistent with our hypothesis that the resistant cells have undergone a general shift in gene expression.

Cell death/proliferation roles for nc886, a non-coding RNA, in the Protein Kinase R pathway in cholangiocarcinoma

We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of protein kinase R (PKR). PKR’s pro-apoptotic role through eukaryotic initiation factor 2 α (eIF2α) phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially, we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR’s activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival nuclear factor-κB pathway. Thus, PKR has a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886’s role in the PKR pathway during tumorigenesis.

Converting cell lines representing hematological malignancies from glucocorticoid-resistant to glucocorticoid-sensitive: Signaling pathway interactions

Mitogen-activated protein kinases (MAPKs), protein kinase A (PKA) and mTOR pathways modulate the apoptotic effects of glucocorticoids (GCs) in human lymphoblastic leukemia CEM cells. We now show that manipulation of these pathways converts several cell lines, representing other lymphoid malignancies, from GC-resistant to GC-sensitive. Basal levels of phosphorylated JNK and ERK were elevated in the GC-resistant cells. Treatments that directly or indirectly reduced phosphorylated JNK and ERK resulted in Dex sensitivity in five resistant lymphoid cell lines. Sensitivity to GC-driven apoptosis correlated with GC-dependent increases in phosphorylated and total glucocorticoid receptor, and in increased levels of the pro-apoptotic protein Bim.

Oxysterol sensitive and resistant lymphoid cells: correlation with regulation of cellular nucleic acid binding protein mRNA.

Oxygenated derivatives of cholesterol inhibit cholesterol synthesis, prevent lymphoid cell growth, and evoke cell death. We have employed a novel selection method to isolate M10 cells, a line of oxysterol-resistant cells, from the sensitive clone CEM C7. Concentrations of the potent sterol 25-hydroxycholesterol that occupy the oxysterol binding protein cause cell death in CEM C7, but not in M10 cells. Both cell lines have similar amounts of the oxysterol binding protein with similar affinities for oxysterol. However, in neither line are the levels of oxysterol binding protein mRNA affected by 1 microM 25-hydroxycholesterol. Furthermore, both cells express the cellular nucleic acid binding protein (CNBP), a 7 zinc finger, DNA-binding protein of unknown function, regulated by oxysterols. The levels of CNBP mRNA are significantly reduced by 25-hydroxycholesterol in the sensitive CEM C7 cells, in which the dose response and time course are consistent with occupancy of the oxysterol binding protein by oxysterol and with subsequent cell kill. However, in the resistant M10 cells, CNBP mRNA levels are unaffected by these concentrations of the 25-hydroxycholesterol. Our results suggest a role for CNBP in oxysterol-induced regulation of cell viability and growth.