ANÁLISIS DE COSTO DEL SISTEMA DE CONEXIÓN A RED 2.4 KWP,

1.5.2.1 MGMT

The DNA repair enzyme MGMT also known as O6-alkylguanine-DNA alkyltransferase (AGT), plays an important role in resistance to TMZ and other alkylating agents. The chemoresistant activity of MGMT has been shown in several cell culture and xenograft studies. The DNA damage induced by addition of methylated adducts by TMZ and other alkylating agents can undergo single-step error free reversal in the presence of MGMT (Esteller et al., 2000). MGMT forms a covalent bond between the alkylated bases and an internal cysteine residue within its active site. It then directly transfers the alkyl group from the DNA to the cysteine thereby restoring the structural integrity of methylated bases in the DNA (Payne et al., 2005). Thus, MGMT can confer resistance to the cytotoxicity induced by alkylating agents and increased MGMT expression is associated with in vitro and in vivo GBM resistance to first line drug TMZ, other nitrosoureas and alkylating drugs (Dolan et al., 1990). However, in this process, MGMT undergoes self degradation through the ubiquitin-proteasome pathway after binding alkyl groups from DNA and hence the cells have to re-synthesize MGMT molecule (Chen et al., 1998; Esteller and Herman, 2004). Research has shown that the expression levels of MGMT are not the same in all patients and differ with individuals as well as with the grade and aggressiveness of the tumour. Epigenetic inactivation of MGMT gene by promoter hypermethylation results in low or no MGMT expression and lowers cellular ability to carry out DNA repair (Esteller and Herman, 2004). Hence, GBM patients lacking tumour MGMT expression benefit from treatment with

23 alkylating drugs and show prolonged survival compared to MGMT-positive GBM patients. For example in the case of first line drug TMZ, a median survival of 21.7 months was seen among patients with a methylated promoter who received TMZ + radiotherapy compared to patients with a unmethylated promoter who had a median survival of just 12.7 months with the same treatment. Therefore, MGMT promoter methylation status has been shown to predict clinical response to alkylating agents (Hegi et al., 2005). The above facts suggested that inactivation of MGMT using inhibitors could improve treatment outcomes and made MGMT a suitable target for intervention. An inhibitor like O6-benzylguanine (O6-BG) that irreversibly inactivates MGMT is shown to enhance BCNU and TMZ cytotoxicity in MGMT-positive glioma cells both in vitro and in vivo but the therapeutic potential of adding O6-BG to TMZ-treatment has so far also been discouraging (Jaeckle et al., 1998).

1.5.2.2 DNA Mismatch repair

The second important mechanism of resistance against chemotherapy is the DNA mismatch repair mechanism (MMR). MMR is a normal DNA repair pathway that maintains genomic integrity by correcting replication errors, which escape the DNA polymerase proof reading process (Hickman and Samson, 1999). MMR acts as direct sensors to DNA damage signalling through ATR checkpoint and this property is therefore considered as an important tumour suppressor mechanism. Absence of a functional MMR system due to mutations in the protein complexes results in unnoted drug-induced DNA damage that contribute to further mutagenesis and reduced anti-tumour activity of

24 methylating anticancer drugs (Fink et al., 1998). For example in the case of TMZ treatment DNA polymerase mispairs O6-meG with thymine (instead of C opposite) and a normal MMR system should recognise base pair by MutS a heterodimeric complex made of two MMR proteins HMSH2 and HMSH6. This in turn will interact with MutL to excise the damaged strand of DNA. However, O6- meG remains and binds with another thymine thereby activating multiple cycles of MMR. Futile cycling of MMR, continuous DNA re-synthesis and failure to get past O6- meG halts DNA replication and leads to checkpoint activation finally causing breaks in DNA strand (Taverna et al., 2000). Thus, cytotoxicity induced by TMZ largely depends on functional MMR and MMR mutated or deficient cells are shown to be more than 100-fold more resistant to alkylating agents like TMZ.

1.5.2.3 Base excision repair and PARP-1

Endogeneous DNA damage resulting from methylation, deamination, hydroxylation and ROS are repaired by the DNA base excision repair system (BER). The BER pathway also repairs DNA base lesions induced by drugs like TMZ and other alkylating agents (Frosina, 2000). Apart from O6-meG, several other DNA adducts as N7-methylguanine, O3-methyladenine and N3- alkyladenine are produced by drugs. These alkylated adenine or guanine bases are repaired by the BER pathway in two steps. Firstly a lesion specific DNA repair enzyme alkyladenine DNA glycosylase (AAG) removes those bases from the DNA strand leavng single nucleotide gaps known as AP sites since they are either apurinic (devoid of A or G) or apyrimidinic (devoid of C or T). In

25 the second step the AP site is recognised by an AP-endonuclease (APE1) that makes an incision in the strand and fills the single nucleotide gap by DNA polymerase- (pol-)-mediated DNA synthesis and DNA ligase that seals the nick in the strand (Trivedi, 2005; Frosina, 2000). Poly-ADP-ribose polymerase-1 (PARP1) is an important enzyme in the BER pathway with zinc finger DNA binding nuclease activity and acts as a sensor of DNA damage. It facilitates repair of single stranded or double stranded breaks in the DNA by coordinating with other pathways like BER and MMR (de Murcia et al., 1997). Inhibition of PARP is shown to suppress the function of BER pathway and significantly enhance the cytotoxicity of alkylating drugs in GBM cells (Tentori et al., 2002). In addition, it is also shown that most GBM specimens have very high basal levels of PARP expression especially after treatment with TMZ (Wharton et al., 2000; Cheng, 2005). Both BER and PARP expression are correlated to suppression of MMR activity that further worsens chemoresistance.