Hodgkin disease‐derived cell lines expressing ubiquitous mitochondrial creatine kinase show growth inhibition by cyclocreatine treatment independent of apoptosis

Abstract

Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. In Hodgkin disease (HD), the telltale Hodgkin and Reed-Sternberg (H-RS) cells are surrounded by reactive tissue components. The scarcity of the malignant cells has hampered their genetic analysis. Only recently, single cell PCR analysis could show a clonal B-cell origin of the H-RS cells. 1 Sequence analysis demonstrated that these cells lost their immunoglobulin (Ig) expression due to crippling mutations acquired in the germinal centre. Normal B-cells unable to express Ig are prevented from antigenic selection and are eliminated by apoptosis. The transforming event in HD allowing the cells to escape from programmed cell death is not known. One possible candidate is Epstein-Barr virus (EBV), which is found in 50% of HD cases and has been shown to upregulate the antiapoptotic gene bcl-2 in infected cells. 2 In addition, the complex chromosomal aberrations seen in HD 3 may cause changes in gene expression resulting in resistance to apoptosis. Because of the considerable karyotypic variation from case to case, different molecular mechanisms may be involved in malignant transformation in HD and, in support of this, no HD specific genetic aberration could be identified so far.One of the mechanisms possibly involved in malignant transformation is overexpression of creatine kinase (CK). The enzyme reversibly catalyzes the transfer of a phosphate group from ATP to creatine (Cr) and occurs as different isoforms. The cytosolic braintype isoform (B-CK) is coexpressed with the ubiquitous mitochondrial isoform (uMtCK) in many cells and tissues with a high energy demand like brain, placenta, kidney, testis, sperm or endothelial cells. 4 Overexpression of CK has been demonstrated in a wide variety of solid tumors 5-8 and tumor cell lines. 9 Elevated levels of CK have been detected in the sera of patients with advanced-stage cancer and are characteristic for a poor prognosis. 6,8,10 CK catalyzes the reversible transphosphorylation between ATP and creatine: creatine ϩ ATP 7 phosphocreatine ϩ ADP.The enzyme is thought to have 2 main functions in energy metabolism. 4 It buffers the cellular ATP pool by maintaining high cytosolic concentrations of phosphocreatine (PCr), which can be used in times of high cellular energy demand for the regeneration of ATP and it maintains an energy shuttle between subcellular sites of energy supply (oxidative phosphorylation, glycolysis) and sites of ene...

Progressive decrease of phosphocreatine, creatine and creatine kinase in skeletal muscle upon transformation to sarcoma

“…Immunoblot experiments with antibodies against uMitCK indicate overexpression of this isoform in gastric and colonic adenocarcinoma. Similar overexpression of uMitCK had been observed in different tumor cell lines [21,22].…”

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle

“…Secondary antibodies goat anti-mouse Cy5 and goat anti-rabbit Cy3 were from Amersham Biosciences, goat antirabbit FITC and monkey anti-sheep FITC were from Pierce, and goat anti-rabbit horseradish peroxidase (HRP) was from Nordic (Tilburg, The Netherlands). Primary antibodies: monoclonal mouse anti-voltage-dependent anion channel (VDAC) was from Calbiochem (USA), COOH-and NH2-terminal polyclonal CRT antipeptide antibodies were produced as described (20), polyclonal adenine nucleotide translocase (ANT) antipeptide antibodies were produced as described (50), mouse anti-cytochrome c oxidase subunit IV (COX) was from Molecular Probes, polyclonal sheep anti-CRT antibodies were produced by ANAWA (Wangen, Switzerland), and their specificity against CRT protein has been established (unpublished observations), polyclonal anti-human sarcomeric mitochondrial creatine kinase (Mis-CK) antibody (32) and polyclonal anti-human cytosolic muscle-type (M)-CK antibody (54) have been described.…”

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

“…This intimate exchange of substrates and products, the so-called functional coupling or metabolite channeling (10,11), fulfills important functions that may vary among different tissues, species, and developmental states (12,13): (i) phosphocreatine becomes the high energy intermediate that is exported from mitochondria into the cytosol (3); (ii) locally generated ADP stimulates oxidative phosphorylation (11); and (iii) ADP channeled through the MtCK/ANT interaction inhibits the Ca 2ϩ -induced opening of the mitochondrial permeability transition pore (14,15), a well known trigger of apoptosis (16,17). Thus, overexpression of uMtCK in many malignant cancers with especially poor prognosis (18,19) may be related to high energy turnover and failure to eliminate cancer cells via apoptosis. MtCK functions in energy buffering and transport, as well as permeability transition pore regulation may also explain the supportive and protective effects of the CK substrate creatine in many muscular, neurodegenerative, and age-related disorders (20 -22).…”