Identification of novel human tumor cell-specific CaMK-II variants

AbstractCaMK-II (the (type II) multifunctional Ca2+/CaM-dependent protein kinase) has been implicated in diverse neuronal and non-neuronal functions, including cell growth control. CaMKII expression was evaluated in a variety of human tumor cell lines using RT-PCR (reverse transcriptase coupled polymerase chain reaction). PCR primers which flanked the CaMK-II variable domain were used so that all possible variants of the four mammalian CaMK-II genes (α, β, γ and δ) could be identified. 8 distinct CaMK-II isozymes were identified from human mammary tumor and neuroblastoma cell cDNA, each of which represented a variant of β, γ or δ CaMK-II. They included 2 β isozymes (βe, βe′), 4 γ isozymes (γB, γC, γG, γH) and 2 δ isozymes (δC, δE) This is the first report of human β and δ CaMK-II sequences.A panel of human cell types was then screened for these CaMK-II isozymes. As expected, cerebral cortex predominately expressed α, β and δA CaMK-II. In contrast, tumor cells, including those of neuronal origin, expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. Tumor cells of diverse tissue origin uniformly lacked α CaMK-II and expressed 1–2 β isozymes, at least 3 γ isozymes and 1–2 δ isozymes. When compared to undifferentiated fibroblasts, βe, βe′, γG and γH were preferentially expressed in tumor cells. CaMK-II immunoblots also indicated that neuroblastoma and mammary tumor cells express isozymes of CaMK-II not present in their non-transformed cell or tissue counterpart. The identification of these new, potential tumor-specific CaMK-II variants supports previous indications that CaMK-II plays a role in growth control. In addition, these results provide insight into both splice variant switching and variable domain structural similarities among all CaMK-II isozymes

Zebrafish Model of MLL-Rearranged Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of leukemia. Standard treatment includes chemotherapy as well as stem cell transplantation, but for aging patients and those with impaired immune function these rigorous therapies are not always possible. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) exhibit far worse prognoses than patients without. Given these circumstances new therapies must be developed. Methods: Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. We developed a transient transgenic model exhibiting AML characteristics by microinjecting single-cell zebrafish embryos with a tissue specific MLL-ENL expression construct. Results: We found that the expression of MLL-ENL induced a clustered expansion of MLL+ and pu.1+ myeloid cells on the yolk sac at 48 and 72 hours post fertilization (hpf). To characterize our transient AML model, we treated MLL-ENL expressing embryos with either one of or a combination of two drugs that are currently being used in human AML drug trials, Venetoclax and Flavopiridol. We found that treatment with either drug reduced the myeloid expansion induced by the expression of MLL-ENL, and that co-treatment reduced the observed myeloid expansion even further. Conclusions: Although further analysis is required, these data suggest that we successfully developed a transient transgenic AML model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually.https://scholarscompass.vcu.edu/gradposters/1112/thumbnail.jp

α-Adrenergic inhibition of proliferation in HepG2 cells stably transfected with the α1B-adrenergic receptor through a p42MAP kinase/p21Cip1/WAF1-dependent pathway

AbstractActivation of α1B adrenergic receptors (α1BAR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of α1BAR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express α1BAR, were stably transfected with a rat α1BAR cDNA (TFG2 cells), in order to study the effects of maintained α1BAR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of α1BAR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAP kinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEK1 inhibitor PD98059, or infection with a −/− MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 −/− blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of α1BARs in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via α1BAR appears to be p42MAP kinase and p21Cip1/WAF1 dependent

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization

Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish.

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression