1.8.1 Nucleoside diphosphate kinases
Nucleoside diphosphate kinases (NDPKs) are a family of highly conserved proteins that have multifunctional purpose in mammalian cells. There are nine human NDPK isoforms, which are transcribed from different nm23 genes. The most widely expressed isoforms are NDPK-A and NDPK-B. These isoforms act as cellular nucleotide converters and regulate the energy currency in the cells, adenosine triphosphate (ATP) (Jovanovich, et al., 2007).
ATP + GDP ADP + GTP
NDPK-A catalyses the production of ATP in mammalian cells via gamma-phosphate transfer between nucleoside diphosphates to nucleoside triphosphates. However NDPK-A does not work
alone. The adenosine monophosphate (AMP) – activated protein kinase (AMPK) is a
heterotrimeric protein complex that responds to cellular energy status (Hardie, et al., 1999). If ATP becomes scarce in the cell, the resulting rise in cellular AMP levels activates AMPK (Hardie,
et al., 1997). AMPK phosphorylates downstream substrates and has a net effect of switching off ATP-utilising pathways such as fatty acid synthesis, and cholesterol synthesis, and turns on ATP- generating pathways such as fatty acid oxidation and glycolysis (Dr Elaine Campbell, Bute Medical School, personal communication).
It has been reported that NDPK-A only (not NDPK-B) selectively regulates the alpha 1 isoform of AMPK independently of AMP concentration (Hardie, et al., 1999). AMPK alpha 1 uses the ATP generated by NDPK-A to phosphorylate a recognised downstream in vivo target, acetyl coenzyme A carboxylase (ACC).Once ACC is phosphorylated it promotes ATP conservation by
NDPK-B
inhibition of fatty acid synthesis and encourages ATP-generating pathways such as fatty acid oxidation (Campbell, personal communication).
The other side of the coin, NDPK-B equilibrium favours using ATP to generate guanosine triphosphate (GTP) to supply heterotrimeric G-protein functions (Hippe, et al., 2006). This process
of ATP production and conservation via NDPK-A and NDPK-B is regulated by Protein Kinase CK2 formally known as Casein Kinase 2 (CK2), (Jovanovich, et al., 2007). CK2 is a constitutively active two alpha and two beta heterodimer and has been found to have an essential role in almost every process in mammalian cells (Jovanovich, et al., 2007).
1.8.2 Casein kinase 2 (CK2)
When cells are unstressed, CK2 alpha is bound to NDPK-A and AMPK and has an inhibitory effect on NDPK-A so that it does not generate ATP local to AMPK and therefore ACC is active and ATP consuming pathways are switched on e.g. fatty acid synthesis. As a result, CK2 is not bound to NDPK-B which functions as normal with ATP being used to generate GTP and G- protein pathways are active (Jovanovich, et al., 2007; Campbell, personal communication). Oxygen tension or stress can affect this process and causes CK2 to translocate to NDPK-B. This results in NDPK-A being free to supply AMPK with ATP which results in ATP generating pathways being switched on via AMPK phosphorylation (Campbell, personal communication). NDPK-B is thus inhibited by CK2 and no longer uses ATP to generate GTP and hence G-protein pathways are repressed.
1.8.3 Lactate dehydrogenase (LDH)
Campbell, personal communication). LDH is a tetramer composed of two isoforms A or B and there are five human isoenzymes (Kaplan, 1963). LDH-1 is found in the heart and is composed of 4 B subunits and is known as LDH-B. LDH-2 is located in reticuloendothelial system cells. LDH-3 is expressed in the lungs, and LDH-4 has been discovered in the kidneys. LDH-5 is located in the liver and striated muscle and is composed of 4 A subunits and named LDH-A. LDH-A has the highest efficiency to catalyse pyruvate to lactate, particularly under hypoxic conditions (Goldman,
et al., 1964).
Under normal conditions, LDH-B is bound to NDPK-B which functions normally as discussed. However if LDH-A is bound to NDPK-B it augments CK2s inhibitory effect as described. Thus, LDH-A acts as a regulatory subunit of NDPK-B s GTP output and ATP generation (Jovanovich, et al., 2007).
Glycolysis is sensitive to the oxidative stress of the cells. In aerobic conditions, pyruvate and nicotinamide adenine dinucleotide in its reduced form (NADH) are used via oxidative phosphorylation. In hypoxic conditions lactate and nicotinamide adenine dinucleotide oxidised form (NAD+) are used via continuous anaerobic glycolysis.
Pyruvate + NADH Lactate + NAD+
Therefore, if oxygen is in plentiful supply, LDH-B is expressed in the cells and is bound to NDPK- B. However if oxygen is scarce, LDH-A preferentially binds to NDPK-B, augmenting CK2 alpha which has an inhibitory effect on NDPK-Bs ability to make GTP from ATP and G-protein processes are depressed to conserve ATP.
LDH-A LDH-B Aerobic Hypoxic Oxidative phosphorylation Continuous anaerobic glycolysis
Cancer cells express LDH-A independent of the oxygen concentration available to the cell and produces energy via continuous anaerobic glycolysis. This is known as the Warburg Effect (Warburg, 1956).
1.8.4 c-Myc
A novel sting in the tail is the proto- oncogene c-Myc. It has been shown that c-Myc is bound to the NDPK-B complex in the cytoplasm of cells under normal conditions. When it is bound it is inactive, however when cells get stressed and LDH-A /CK2 alpha binds to NDPK-B, c-Myc is ejected from the complex, (Campbell, personal communication). It may translocate to the nucleus as nuclear downstream targets of c-Myc are noted to be up regulated, (Campbell, personal communication).
c-Myc is a transcription factor and has three closely related members in its family; c-Myc, L-Myc, and N-Myc (Williams, et al., 2005). They each have very distinct patterns of expression but can compensate if one of the isoforms is lost (Williams, et al., 2005). Myc forms a heterodimeric transcription factor with its partner Max (Blackwood, et al., 1991). Myc expression regulates cell proliferation (by activating cyclins), differentiation, and apoptosis (Williams, et al., 2005). Myc controls various cell cycle activities including regulating cell cycle checkpoints by binding to and activating cyclins. This includes cyclin D2 in which activation corresponds to pushing the cell into S phase. Myc is also indirectly involved in reducing expression of protein pathways, such a p21WARF1 and p15INK4b which are involved in cell cycle arrest (Williams, et al., 2005).
Over expression of c-Myc has been observed in prostate cancer and is directly related to amplification in the 8q24 region as this leads to an increase in copy number of the c-Myc gene (Williams, et al., 2005; Yang, et al., 2005). This suggests that c-Myc in involved with the
models resulted in prostate carcinogenesis (Williams, et al., 2005). The mice models also exhibited a decreased expression of PTEN, a tumour suppressor gene (Williams, et al., 2005). Epidemiological evidence shows that human prostate cancer is associated with genetic variation at chromosome 8q24 in African-American men (Sole, et al., 2008), where it is associated with increase risk of contracting the disease (Freedman, et al., 2006). Mutations which disrupt the regulation or expression levels of c-Myc gene are among the most common found in human and animal cancer (Cole, et al., 1999). Studies have shown transgenic mice with unregulated c-Myc
gene developing prostate neoplasia (Zhang, et al., 2000) and shown that these mice tumours share molecular features with human prostate tumours (Ellwood-Yen, et al., 2003).