Limitless replicative Self-sufficiency in growth signals Sustained angiogenesis Insensitivity to anti-growth signals Limitless replicative potential Evading apoptosis Tissue invasion & metastasis Cancer The development of human cancer cells

Cdc6 is essential in the initiation of DNA replication: A). Assembling pre-replication complex (pre-RC) with ORC, and loading MCMs to replication origins; B). Licensing DNA replication to ensure one round DNA replication per cell cycle.

the specific genes for cell proliferation: The novel function of GLS101/TSUKUSI FL1 is the silencing of human Cdc6, the specific genes for cell proliferation: Specific Cdc6 shRNAs produced from the viral vector Inhibit Cdc6 and block Cancer cell proliferation, but not normal cells. Cdc6 THTD6Ai THTP CHLA255 LA-N-2 Mock MCF7 -actin 1 2 3 4 5 6 7 8 Cdc6 knockdown Cdc6 shRNA

Apoptosis Differentiation Senescence The Cell Cycle M G1 Recombinant Lentivirus GLS101 G1 S G2 M STOP Senescence The Cell Cycle

Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown 1 2 3 4 Phosph-Rb-C Ser780 Phosph-Rb-C Ser807/811 IgG, light chain —    IP/β-p16INK4a — —   THTD  — — — IP/mouse IgG Phosph Rb-C Ser 807/811 1 2 3 4 1:5 1:10 1:25  IP/β-p16INK4a — — —  THTD

Senescence-associated β-glactosidase (SA-β-Gla) positive staining caused by Cdc6 knockdown MCF7 MCF7/THTP MCF7/THTD6Ai

The formation of senescence associated heterochromatic foci via Cdc6 knockdown Anti-H3K9me3/me2 indirect fluorescent staining THTD6Ai Mock Confocal fluorescent microscopy