Self-sufficiency in growth signals

Limitless replicative potential

Sustained angiogenesis

Evading apoptosis

Insensitivity to anti-growth signals

Tissue invasion & metastasis

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 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 blockCancer cell proliferation, but not normal cells.

Cdc6 knockdown

Cdc6 shRNA

Cdc6

TH

TD

6A

i

TH

TP

CHLA255LA-N-2

TH

TP

TH

TD

6A

i

Mo

ck

Mo

ck

TH

TP

TH

TD

6A

i

MCF7

-actin

1 2 3 4 5 6 7 8

G1

S

G2

M

STOP

Recombinant Lentivirus

GLS101

1 2 3 4

Phosph-Rb-C Ser780

Phosph-Rb-C Ser807/811

IgG, light chain

— IP/β-p16INK4a

— — THTD — — — IP/mouse IgG

IgG, light chain

Phosph Rb-C Ser 807/811

1 2 3 4

1:5

1:10

1:25

IP/β-p16INK4a

— — — THTD

Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown

MCF7

MCF7/THTP

MCF7/THTD6Ai

Senescence-associated β-glactosidase (SA-β-Gla) positive staining caused by Cdc6 knockdown

Anti-H3K9me3/me2indirect fluorescentstaining

THTD6AiMock

Mock THTD6Ai

Confocal fluorescent microscopy

The formation of senescence associated heterochromatic foci viaCdc6 knockdown