X-Message-Number: 25488
Date: Sat, 8 Jan 2005 05:26:12 -0800 (PST)
From: Doug Skrecky <>
Subject: p16(INK4a)-specific siRNA fights aging

Rheumatology (Oxford). 2004 May;43(5):555-68. Epub 2004 Mar 16
Recovery of function in osteoarthritic chondrocytes induced by
p16INK4a-specific siRNA in vitro.
  OBJECTIVE: To demonstrate the roles of p16(INK4a) in the senescence of
human chondrocytes and the progression of osteoarthritis (OA). METHODS:
Immunohistochemistry and reverse transcriptase polymerase chain reaction
(RT-PCR) were performed to examine p16(INK4a) expression in fetal, normal
age-matched and OA cartilage, and Western blot was used in primary
cultured chondrocytes from different origins. To explore a functional
p16(INK4a) knockdown in OA chondrocytes, the primary cultured cells were
treated with p16(INK4a)-specific small interfering ribonucleic acids
(siRNAs). Expression of p16(INK4a), p14(ARF) and p53 was observed by
Western blot and RT-PCR. The phosphorylation status of pRb,
senescence-associated beta-galactosidase (SA-beta-gal), cell G1/S
transition and cell proliferation were studied by Western blot,
histological staining, 3H-thymidine incorporation and cell counts
respectively. Expression of the collagen I, collagen II and aggrecan
genes was measured by semiquantitative RT-PCR. To establish the response
of chondrocytes to cytokines, cells were treated with transforming growth
factor-beta1 (TGF-beta1) or interleukin-1alpha (IL-1alpha) and examined
for incorporation of 3H-thymidine, 3H-proline and 35S-sulphate
respectively. RESULTS: A significant increase of p16(INK4a) was detected
in OA chondrocytes compared with normal age-matched and fetal
chondrocytes (P<0.01) in vivo and in vitro. Treated with
p16(INK4a)-specific siRNAs, OA chondrocytes displayed a significant
decrease in p16(INK4a) expression with an increase of phosphorylated pRb,
but no alteration of p14(ARF) and p53 expression, followed by decreases
of senescent features and increases in the expression of some
chondrocyte-specific genes and overall repair capacity. CONCLUSIONS:
p16(INK4a) is instrumental in the senescence of human articular
chondrocytes or OA. The reduction of p16(INK4a) by RNA interference
(RNAi) contributed to the recovery of osteoarthritic chondrocytes,
suggesting that p16(INK4a) may be a viable future therapeutic candidate.

Exp Cell Res. 2004 Jan 1;292(1):151-6
Direct evidence from siRNA-directed "knock down" that p16(INK4a) is
required for human fibroblast senescence and for limiting ras-induced
epithelial cell proliferation.
  The selective pressure for disruption of the cyclin-dependent kinase
inhibitor p16(INK4a) in human cancer has been postulated to reflect its
role in mediating growth arrest, both in response to telomere erosion
(replicative senescence) and to oncogene-induced and other "stress"
signals. Given the known species-specific differences in regulation of
senescence, we have tested this hypothesis in human, as opposed to
rodent, cells by designing a small interfering RNA (siRNA) to knock down
p16(INK4a) expression. Transfection of this siRNA into late-passage
normal human diploid fibroblasts allowed at least temporary escape from
entry into replicative senescence. Furthermore, in our in vitro model of
early-stage, RAS-induced thyroid tumorigenesis, sequential transfections
with this siRNA allowed outgrowth of small clusters of proliferating
epithelial cells, consistent with escape from the spontaneous
"senescence", which normally curtails their proliferative response to
mutant RAS. These data provide the first direct evidence that p16(INK4a)
is necessary for the initiation of both telomere-dependent and
telomere-independent senescence in human cells.

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