Investigating the genetic instability and expression of four DNA repair related proteins in the peripheral lymphocytes of 36 untreated lung cancer patients Medical school of Zhejiang University He Jiliang

Genetic Variability

Roles 0f DNA damage,DNA repair, Cell cycle arrest and apoptosis in carcinogenesis

Genetic instability of cancer  One of the main causes of cancer is high genetic change or genetic instability.  Genetic instability is a transient or a persistent state that causes a series of mutational events leading to gross genetic alterations.  It is now clear that most cancers have altered genomes, and genetic instability has been found in many types of cancers.  The question whether genetic instability is a cause or a consequence of tumorigenesis has been debated for years.

The term “genetic instability” is generic : nucleotide alteration genomic instability (at the gene level) (chromosomal level ) faulty or leaky (a) microsatellite instability DNA repair pathways (MIN) (b) chromosomal instabilities (CIN) in chromosome structure and number Genetic instability

Genetic instability of cancer (A)DNA replication mechanisms : (1)DNA repair pathways: keeping genes and genome intact (2)chromatin epigenetic modification pathways; (B) maintenance of mitotic stability and chromosome integrity DNA repair pathways safeguard the genome from deleterious mutations Mechanisms maintaining chromosome integrity

(a) nucleotide excision repair (NER); (b) base excision repair (BER); (c) homologous recombination repair (HRR); (d) non-homologous end joining (NHEJ); (e) mismatch repair (MMR) Main DNA repair mechanisms

Tumor suppressor genes (targets for many genetic changes) gatekeeper genes caretaker genes act directly to regulate do not directly regulate cell proliferation proliferation but regulate cell-cycle checkpoints, DNA repair and apoptosis Tumor suppressor genes

 ATM gene is a caretaker gene ( a checkpoint gene).  ATM protein is a large serine/threonine protein kinase and has more than 15 known substrates.  After activation by ionizing radiation, ATM kinase initiates a cascade of signaling pathways and culminates in cell cycle arrest, apoptosis and DNA repair.  ATM plays a key role in safeguarding genome integrity. ATM gene and ATM protein

The aims of present study Chromosomal instability may be one of the primary causes of tumor cell evasion of therapy, understanding of biologic basis of chromosomal instability is critical for effective diagnostic and prognostic evaluation and therapeutic intervention of cancer.

(A) To observe whether the genetic instability occurs in peripheral lymphocytes of lung cancer patients with MN assay and comet assay. (B) To observe the genetic instability is associated with four DNA repair related proteins (ATM, DNA ligase Ⅲ, DNA ligase Ⅳ and XPF) in lung cancer patients As markers of susceptibility were measured using west blotting.

1.The blood samples (1) 36 untreated lung cancer patients (15 females and 21 males, mean age years old) (2) 30 controls (16 females and 14 males, mean age years old) (3) Each sample was divided into two parts: (a) irradiated sample (exposed to 3 Gy X-ray) (b) non-irradiated sample (not exposed to 3 Gy X-ray) Materials and Methods

2. Detecting Methods (1) Chromosomal Damage Micronucleus (MN) assay can be used to detect (chromosome loss and chromosome breakage) (2) DNA Damage Comet assay can be used to detect (various forms of DNA damage ) (3) Protein expression West blotting was used to detected: ATM, XPF, Ligase Ⅲ and Ligase Ⅳ Materials and Methods

Blood Samples Part 1 Part 2 (Irradiated samples) (non-irradiated samples) Comet assay MN assay West blotting Chromosomal Damage DNA Damage Protein expression

Results 1 (comet assay) PatientsControls Non-irradiated Samples irradiated Samples Non- irradiated Samples irradiated Samples MTL (μm) 1.88 ± ± ± ± 0.11 MTM 0.84 ± 0.07 b 1.09±0.11 b 0.60 ± ± 0.10

Fig.1. Spontaneous (MTL0 and MTM0) and IR-induced (IR-MTL and IR- MTM) genetic damage in lung cancer patients and controls was detected by comet assay. MTL=mean tail length, MTM=mean tail moment, IR=ionizing radiation, * P <0.05.

Results 2 (MN assay) PatientsControls Non-irradiated Samples irradiated Samples Non-irradiated Samples irradiated Samples MCF (‰ ) 9.25±0.58 b 66.14±2.07 a 6.10± ±1.71 MNF (‰ ) 10.17±0.72 b 75.64±2.34 a 6.60± ±2.13

Fig.2. Spontaneous (MCF0 and MNF0) and IR-induced (IR-MCF and IR- MNF) genetic damage in lung cancer patients and controls was detected by MN assay. MCF=micronucleated cell frequency, MNF=micronucleus frequency, IR=ionizing radiation, * P <0.05.

Results 3 (West blotting) ATM Ligase Ⅲ Ligase Ⅳ XPF Patients 0.75±0.08 a 0.59±0.07 a 0.54±0.05 a 0.84±0.07 Controls 1.63± ± ± ±0.21

Fig.3. Four DNA repair related proteins expression levels in DNA of lymphocytes collected from lung cancer patients and controls were measured by west blotting. * P <0.05.

Fig.1 shows: Four DNA related proteins and loading control (GAPDH) Results 4

Fig.4.Four DNA related proteins and loading control (GAPDH) were detected by west blotting. A: ATM protein, B: Ligase Ⅲ protein ， C: Ligase Ⅳ protein ， D: XPF protein. No. 1, 3 were from control group, No. 2, 4 were from lung cancer patient group

(1) The genetic instability in peripheral lymphocytes of 36 lung cancer patients was significantly higher than that of controls (2) The increased genetic instability in peripheral lymphocytes of lung cancer patients may be associated with the reduced expression of DNA repair related proteins. Conclusion

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