The work presented here is the first to causally link down-regulated DNA MMR gene expression with the initiation and development of MCTS in-vitro. We used silencing RNA (siRNA) to knockdown expression of the MMR genes MLH1 and PMS2 in surface-associated N2a and CHLA-02-ATRT cultures. Our data show that silencing of these two genes results in an increase in the number and size of MCTS that was observed over several subsequent passages following transfection. The use of both murine and human tumour cell lines eliminates the possibility of the observations being species-specific.
Several possibilities exist to explain why impaired DNA MMR enhances MCTS growth. MMR proteins serve to correct post-replication errors by eliminating base-base mismatches and insertion or deletion loops. An increase in spontaneous mutation frequency resulting from an impairment of this system may give rise to a range of beneficial and deleterious mutations. Such a mechanism has been demonstrated by Nicolaides et al 
who, in an alternative approach to siRNA performed transfection studies using a naturally occurring mutant hPMS2
identified from the germline of a HNPCC patient. The nonsense mutation was sufficient to suppress DNA MMR pathways and induce microsatellite instability in cells containing wild-type hPMS2
. Mutator phenotypes of this kind represent an important model for cancer development whereby mutant cells selected for their ability to proliferate, while surviving environmental stresses, expand their numbers and contribute to MCTS growth 
. This hypothesis is supported by data in this paper which reports that spheroid-derived cells exhibit a higher propensity to form MCTS compared to cells derived from the 2-D monolayer
However, MMR proteins have several functions other than direct DNA repair that are relevant to genomic stability and carcinogenesis. These include DNA damage surveillance, damage signaling and apoptosis 
, prevention of recombination between non-identical sequences 
and participation in meiotic processes (chromosome pairing) 
. In addition there is now evidence that the MMR system is integral to the signaling processes that activate cell-cycle checkpoints or apoptosis. Following DNA damage, cell cycle checkpoint activation halts the cell cycle allowing time for DNA repair. Repair occurs in the G1-S and G2-M phases. Previous studies have shown that defective expression of MLH1
alters G2-M cell cycle checkpoint control in human colon carcinoma cells exposed to ionizing radiation 
. Similar observations have been made in MLH1
deficient mice. Further work has suggested that MMR complexes play a key role in linking Ataxia telangiectasia mutated (ATM) and the downstream effector, CHK2 checkpoint homolog (CHEK2), in response to DNA damage. CHK2 activation is lost in cells deficient in MMR causing the G1-S checkpoint to fail. Such interactions suggest a relationship between checkpoint signaling activated by DNA damage and mechanisms of DNA repair. In addition to the mutator phenotype associated with deficiency in MMR, abrogated S-phase checkpoint activation may further explain the genomic instability and cancer predisposition arising from inactivation of the MMR system.
It must also be stated that the MMR system can mediate cytotoxicity. Down-regulation of MMR has been directly implicated in the resistance of cancers to radiation and a number of chemotherapeutic agents 
. Impaired MMR results in drug resistance through two mechanisms: the inability to detect DNA damage and subsequent activation of apoptosis and through and increase in mutation rate throughout the genome 
. Further studies to explore the clinical significance of MMR deficient cells in tumours are needed.
Previous data has also suggested that human neuroblastoma cell lines contain pluripotent tumour-initiating cells or ‘cancer stem cells’ distinguished by CD133 (a neural stem cell marker) expression 
. Cancer stem cells have been seen to comprise the majority of a tumour mass before terminal differentiation 
. Current models of cancer development imply that genetic instability and thus mutations directly affect normal stem cells and their cellular pathways causing them to become malignant 
. In neuroblastoma CD133 positive cells show enhanced MCTS formation 
. It is therefore possible that down-regulated DNA MMR may contribute to the genetic instability in stem cells, promoting MCTS initiation and growth.
Consistent with reports on numerous other human and murine cell lines grown as spheroids 
we have demonstrated down-regulation of MLH1
expression in N2a and CHLA-02-ATRT MCTS compared with non-spheroid forming ‘monolayer cells’. Recent evidence indicates that the expression of key DNA MMR genes is down-regulated under conditions of hypoxic stress. Hypoxia is a common feature of the tumour microenvironment, arising from imbalances between oxygen supply and consumption. Emerging evidence suggests that hypoxia-induced genetic instability is a key mechanism underlying tumour propagation. Reynolds et al. 
and more recently Papp-Szabo et al. 
have reported increased mutation frequencies in cells exposed to hypoxia-reoxygenation cycles. Further studies have implicated hypoxia in oxidative base damage 
, gene amplification 
and DNA over-replication 
. Importantly for this present study hypoxia has been implicated in the repression of nucleotide MMR pathways and MLH1
down-regulation is specifically enhanced under conditions of hypoxic stress 
. Koshiji et al 
have since demonstrated the down-regulation of MSH2 and MSH6 in response to hypoxia. Further alterations in the expression and activation patterns of numerous DNA repair and stress-response factors have been detected, however, the mechanisms responsible for these observations remains to be determined. Further work in our laboratory will examine the interrelationship between hypoxia and genetic instability in the context of MCTS initiation and development.
In summary our results show a deficiency of DNA MMR gene expression in murine N2a and human CHLA-02-ATRT cells grown as MCTS in comparison with 2D monolayer cultures. We have shown that down-regulated DNA MMR results in enhanced MCTS initiation and growth in N2a and CHLA-02-ATRT surface-associated culture systems. This shows that down-regulation of MMR can initiate and perpetuate the spheroid microenvironment, which can be an important initiating event in malignancy. Additional work such as an in vitro
DNA repair assay (comparing the ability of cytosolic extracts to repair DNA mismatches 
) could functionally assess the level of DNA mismatch repair activity in tumour spheroids or in the siRNA transfected cells and provide a clearer understanding of the processes that might be responsible for these findings. We believe that by examining distinct MCTS and monolayer subpopulations that arise during surface-culture of N2a and CHLA-02-ATRT, this study has addressed pathways that lead to the initiation and early differentiation of MCTS structures, thus providing new insight into early events in tumourigenesis.