Supplemental Material
Overexpression of cyclin D3 results in increased keratinocyte proliferation
Our studies show a clear regulation of cyclin D2 level by cyclin D3 expression. Thus, we
sought to investigate the proliferative capacity of non-neoplastic keratinocytes derived
from K5-D3 mice. To determine whether the decreased cyclin D2 expression in K5-D3
keratinocytes would result in reduced proliferation, we quantified the number of
nucleated keratinocytes and proliferative cells (BrdU positive) in the interfollicular
epidermis of K5-D3 and wild type sibling mice. Cyclin D3 overexpression results in a
1.5-fold increase in the number of nucleated keratinocytes compared with wild type mice
(P<0.05) without affecting the epidermal thickness (Figure S-1,A and S-1,B). We have
previously reported that cyclin D1 overexpression results in increased sensitivity to TPAinduced proliferation in an in vivo model of normal cell proliferation (Rodriguez-Puebla
et al., 1999). Therefore, we asked whether cyclin D3 would also induce keratinocyte
proliferation or alternatively whether the decreased expression of cyclin D2 would result
in a reduced rate of cell proliferation. In this model one topical application of TPA on the
mice’s back induces a synchronized wave of keratinocyte proliferation that is scored by
immunohistochemical analysis of BrdU positive cells at several time-points after TPA
application (Rodriguez-Puebla et al., 1998). Upon TPA-treatment K5-D3 keratinocytes
enter in S-phase 4 hours earlier than wild type keratinocytes reaching a peak of
proliferation between 16-20 hours (18% of BrdU-positive cells) (Figure S-1,C).
Keratinocytes from wild type mice reached a peak of 12% of BrdU-positive cells
between 20-24 hours (Figure S-1, C). Western blot analysis of K5-D3 epidermis revealed
that cyclin D2 protein levels remained low regardless of the TPA-induced proliferation
(Figure S-1, D). These data indicate that forced expression of cyclin D3 plays a positive
role in TPA-induced keratinocyte proliferation in spite of the reduce level of cyclin D2. It
is worth mentioning that depletion of cyclin D2 reduces by 50% the number of BrdUpositive cells in mouse epidermis compared to normal sibling mice (data not-shown).
Thus, it is formally possible that normal cell proliferation is only affected by cyclin D2
ablation and not by diminished levels of this protein as observed in K5-D3 mice (Figure
S-1, C, time 0).
Collectively, these results demonstrated that reduced expression of cyclin D2 (K5-D3
mice) or complete abrogation of cyclin D2 (cyclin D2-null mice) has an inhibitory effect
in skin tumor development. In contrast, decreased levels of cyclin D2 do not severely
impair the positive effect of cyclin D3 overexpression in nonneoplastic keratinocyte
proliferation.
Increased stability of cyclin D3 protein in non-malignant cell lines
The results presented here demonstrate that cyclin D3 overexpression induces
keratinocyte proliferation, but does not result in advantages in papilloma development.
Indeed, a reduced number of tumors were observed in K5-D3 mice (Figure 2). We have
previously reported that cyclin D1 and cyclin D2 overexpression is associated with Haras activation in mouse papillomas and SCCs whereas cyclin D3 protein levels remained
constant in normal, hyperplastic skin and epidermal tumors (Bianchi et al., 1993; Robles
& Conti, 1995; Rodriguez-Puebla et al., 1998). Consistent with these observations,
ablation of cyclin D1 and cyclin D2 results in a decreased number of papillomas (Robles
et al., 1998) (Figure 4). Thus, we hypothesized that the differences observed in D-type
cyclin protein levels at different stages of tumor development are due, at least in part, to
differences in their stabilities.
To test this hypothesis we used mouse skin tumor cell lines derived from classic
DMBA/TPA protocol that have been characterized as possessing the phenotypes of
different stages of skin neoplasia: C50 (non-tumorigenic), 308 (papilloma) and CH72,
JWF2 (SCCs) (Conti et al., 1988; Fusenig et al., 1983; Ruggeri et al., 1991; Ruggeri et
al., 1994). Cell cultures were treated with cycloheximide, and protein extracts were
analyzed at several time-points to determine the steady-stage of each D-type cyclins.
Correlating well with our previous observations (Bianchi et al., 1993; Robles & Conti,
1995; Rodriguez-Puebla et al., 1998) cyclin D1 remained more stable in carcinoma cell
lines (CH72 and JWF2, half-life 170-147 min) than non-tumorigenic (C50, half-life 75
min) and papillomas (308, half-life 90 min) cell lines (Figure S-2). In contrast, increased
cyclin D3 stability was observed in C50 and 308 cell lines which showed a half life of
160-175 min. Cyclin D2 did not show significant differences in stabilities among these
cell lines.
Altogether, we interpreted these finding as stabilization of cyclin D1 results in selective
advantages during the tumorigenic process whereas cyclin D3 stability does not favor
tumorigenesis. On the other hand, increased stability of cyclin D3 can favor keratinocyte
proliferation, as observed in TPA-induced proliferation (Figure S-1, C). Although,
protein stability of cyclin D2 does not change among the epidermal cell lines studied, it is
evident that the reduction of its protein level decreases the tumorigenic potential of skin
keratinocytes. Therefore, the function of the three members of D-type cyclins appears to
be redundant in normal keratinocyte proliferation, however, each one impinge differently
upon the tumorigenic potential of mouse keratinocytes carrying Ha-ras mutation.
Figure Legends
Figure S-1. Characterization of mouse epidermis from K5-D3 mice. (A) Quantification of
epidermal hyperplasia observed in K5-D3 mice expressed as number of nucleated cells
per millimeter of interfollicular epidermis. Epidermal thickness is expressed in
millimeters. (B) Representative paraffin-sections of dorsal skin from K5-D3 and wild
type mice were stained with hematoxylin and eosine (top panels). BrdU incorporation
was immunoassayed with a monoclonal anti-BrdU antibody (bottom panels). (C) In vivo
kinetic of keratinocyte proliferation expressed as BrdU-positive cells / total number of
cells. Mice were injected with BrdU after TPA treatment for the indicated times. BrdU
labeling was immuno-histochemically detected in paraffin-sections. (D) Western blot
analysis of epidermal lysates from K5-D3 and wild type sibling mice. Cyclin D2 and
cyclin D3 protein level was determined after TPA-treatment. β-actin expression was used
as loading control.
Figure S-2. D-type cyclins protein stability in transformed and non-transformed cell lines.
308 (non-transformed) and JWF2 (transformed) epidermal cell lines were treated with
cycloheximidine and cell extracts were prepared at the indicated times. Protein lysates
were resolved by SDS-PAGE gel and electroblotted onto nitrocellulose and further
immunoblotted with antibodies against cyclin D1, D2 and D3. (C) D-type cyclins
stability was determined by densitometry analysis of the immunoblots. C50 (nontransformed) and CH72 (transformed) cell lines were also used in similar experiments.
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