From the
Adhesion-independent growth is a neoplastic phenotype that is
inducible in Rat 1a fibroblasts by enforced MYC expression.
The c-Myc protein has been well characterized as a transcription
factor, yet the molecular basis of c-Myc-induced neoplastic
transformation has remained elusive. In this report, we demonstrate a
link between ectopic MYC expression, deregulated cyclin A
levels, and adhesion-independent growth.
The MYC protooncogene was known to transform cultured
cells long before it was realized that MYC encodes a
helix-loop-helix-leucine zipper transcription factor,
c-Myc(1, 2, 3, 4, 5) . Yet the
molecular basis for MYC-mediated transformation of cultured
cells has remained poorly understood(6, 7) .
Transformation of primary rat embryo cells by c-Myc is multistep,
requiring the cooperation with an activated RAS gene(3) . c-Myc
alone, however, is capable of transforming a fibroblast cell line, Rat
1a, resulting in anchorage-independent growth(4, 5) .
Based on the recent findings that cyclin A expression is
adhesion-dependent (8, 9) and Myc expression causes
increased cyclin A
levels(10, 11, 12, 13) , we sought to
determine whether Myc-induced adhesion-independent growth is linked to
deregulated cyclin A levels in the Rat 1a cell line. In this report, we
demonstrate that cyclin A links ectopic MYC expression to
adhesion-independent growth.
Soft agarose suspension cultures were performed as
described(11) . Results shown in Fig. 3are for the
Rat1a-InCycA cell line A3 that was described previously(11) .
Another previously characterized (11) inducible cyclin A Rat 1a
cell line A13 is also studied here.
Attached or unattached cells recovered from
culture were counted, and RNAs were harvested using guanidinium
hydrochloride. Poly(A) RNAs were isolated using immobilized oligo(dT).
Randomly primed radiolabeled probes generated from a human c-MYC cDNA were used to detect endogenous rat c-myc mRNA.
Northern analyses were as described(11) .
Ectopic c-Myc expression alters the growth properties of Rat
1a cells. Rat 1a cells expressing ectopic c-Myc (Rat1a-myc) grew faster
and saturated at 25% higher density than mock-transfected Rat 1a cells (Fig. 1A). Rat1a-myc cells proliferate in soft agar
cultures independent of adhesion in contrast to Rat 1a cells, which do
not grow in suspension(4, 5) . We adopted the method of
suspension fibroblast cultures to study the molecular differences
between Rat1a-myc versus Rat 1a cells (Fig. 1B)
(8). Endogenous rat myc mRNA expression was decreased 3-fold
in unattached Rat 1a cells as compared with attached cells suggesting
that endogenous MYC expression is adhesion-dependent (Fig. 2). Rat1a-myc cells expressed transfected human c-Myc
independently of adhesion (Fig. 3A, toprightpanel).
We hypothesized
that c-Myc-induced adhesion-independent growth of Rat1a-myc
cells is mediated through deregulated cyclin A expression, as the
cyclin A level has been linked to c-Myc expression (11-13). In
contrast to the diminished cyclin A level seen in unattached Rat 1a
cells, the cyclin A level remained elevated in unattached Rat1a-myc
cells as compared with attached cells (Fig. 3A). As
observed in Rat 1a cells as well as in NRK and NIH 3T3
fibroblasts(8) , the expression of cyclin D1 and E proteins was
also adhesion-independent in Rat1a-myc fibroblasts. When compared with
Rat 1a cells (Fig. 3A, Rat 1a, lane A3),
confluent Rat1a-myc cells continued to express cyclin A (Fig. 3A, Rat1a-myc, lane A3) and attained
higher saturation density (Fig. 1A). Poly(A)-enriched
RNAs were harvested from Rat 1a or Rat1a-myc cells to determine whether
decreased cyclin A expression in unattached Rat 1a cells occurred at
the mRNA level. Cyclin A mRNA was virtually absent in Rat 1a as
compared with unattached Rat1a-myc cells, whereas cyclin D1 and
glyceraldehyde-3-phosphate dehydrogenase mRNAs were present in both
unattached Rat 1a and Rat1a-myc cells (Fig. 3B). When
considered with previous reports linking MYC expression to
cyclin A levels (11, 12, 13) and cyclin A
expression to adhesion dependence (8), these observations suggest that MYC-induced anchorage-independent growth results from MYC-enforced cyclin A expression. Alternatively, deregulated
cyclin A expression in Rat1a-myc cells does not cause anchorage
independence but is a marker of continued cell proliferation in MYC-induced adhesion-independent
proliferation(15, 16, 17) .
To determine
whether cyclin A expression is able to cause anchorage-independent
growth in Rat 1a fibroblasts, we studied Rat 1a cells (Rat1a-InCycA) in
which cyclin A expression is inducible by zinc through the sheep
metallothionein promoter(11) . We have previously shown that
adherent Rat1a-InCycA cells have a dose-dependent zinc induction of
cyclin A protein expression(11) . Similarly, ectopic expression
of human cyclin A is inducible in the non-adherent Rat1a-InCycA cell
line A3 or A13 as compared with the parental mock-transfected cell line (Fig. 4A). Rat 1a, Rat1a-InCycA (A3), or A13 cells were
grown in soft agarose either in the absence or presence of zinc (Fig. 4B). Rat1a-InCycA (A3) cells formed
adhesion-independent colonies (10
In summary, this report provides insight into the molecular basis of MYC-mediated neoplastic transformation of Rat 1a fibroblasts.
Ectopic MYC protooncogene expression is shown to bypass the
requirement of adhesion for cyclin A expression, whose deregulated
expression allows anchorage-independent cell proliferation.
We thank T. Hunter for cyclin A cDNA, S. Reed for
cyclin E and D1 cDNAs, W. Lee for anti-Myc 9E10 antibody, and J. M.
Roberts for suggestions and reviewing the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cell Cultures and Transfections
Rat1a-myc (pool
RM8) fibroblasts were pooled neomycin-resistant transfectants
expressing human MYC from the Moloney murine leukemia
virus-long terminal repeat promoter(11) . Control Rat 1a
fibroblasts were pooled mock-transfected neomycin-resistant colonies.
Cells were plated at 2 10
/100-mm dish and cultured
in Dulbecco's minimum essential medium with 10% fetal calf serum,
penicillin, and streptomycin. Adherent cells were directly compared
with non-adherent cells, which were prepared in parallel by adding
trypsinized cells in logarithmic growth phase to dishes coated with
0.9% RNase-free agarose as described by Guadagno et al. (8).
Figure 3:
A, human MYC and cyclins
A, D1, and E protein levels in attached (A) or unattached (U) Rat 1a and Rat1a-myc cells were determined by immunoblot
analysis. The number following A or U indicates the time (days) after plating of the cells at 2.0
10
/dish. The samples were loaded after correction
to equalize cell number (10
) per sample. B, mRNA
levels of cyclin A, cyclin D1, or glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) in unattached Rat 1a or Rat1a-myc cells
were determined by Northern blot analysis using equal amounts of
poly(A)-enriched RNA (1 µg/lane). RNAs were harvested at 2
days after cell plating.
Western and Northern Analyses
Western blot
analyses were performed on total cell extracts after SDS-polyacrylamide
gel electrophoresis. Antibodies used were: monoclonal 9E10 anti-human
Myc (described in Hoang et al. (11)); polyclonal rabbit
anti-cyclin A (06-138; UBI, Lake Placid, NY); polyclonal rabbit
anti-cyclin D1 (06-137; UBI); and polyclonal rabbit anti-cyclin E
(06-134; UBI).
Figure 1:
A,
adhesion-dependent growth characteristics of Rat 1a and Rat1a-myc
fibroblasts. Averages of total numbers of cells per 100-mm plate
(duplicates) are shown as a function of time (days) after plating at 2
10
cells/dish. Insets are paired phase
contrast micrographs of Rat 1a (lowerpanel) and
Rat1a-myc (upperpanel) cells at 2 days (leftpair) or 8 days (rightpair) after
plating. B, phase contrast photomicrographs of Rat 1a (left) and Rat1a-myc (right) cells 2 days after
suspension culture over a layer of 0.9% agarose. Unattached cells
prepared this way were directly compared with attached cells in
subsequent experiments.
Figure 2:
Adhesion-dependent expression of
endogenous Rat 1a myc mRNA. Total RNA (20 µg/lane) from
attached (A) and unattached (U) Rat 1a cells was
subjected to Northern analysis using randomly primed radiolabeled human MYC or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes.
Since cyclin A expression was
shown to be adhesion-dependent in NRK(
)and NIH
3T3 fibroblasts(8) , we examined the patterns of cyclin protein
expression in attached versus unattached Rat 1a cells (Fig. 3A)(14) . As compared with attached cells,
unattached Rat 1a cells displayed a severely diminished cyclin A
protein level 1 day after plating, whereas cyclin D1 and E levels were
essentially unaltered. It is also notable that the cyclin A protein
level was markedly diminished in confluent Rat 1a cells (Fig. 3A, Rat 1a, lane A3), indicating that
contact-inhibited growth correlates with the absence of cyclin A. These
observations indicate that cyclin A expression is adhesion-dependent in
Rat 1a fibroblasts, similar to the pattern of expression previously
reported for NRK and NIH 3T3 fibroblasts(8) .
colonies/10
cells plated in agarose) when exposed to zinc, in contrast to Rat
1a cells that did not form large anchorage-independent colonies in
suspension either in the absence or presence of zinc. Another
zinc-inducible cyclin A Rat1a cell line A13 also displayed
anchorage-independent growth with a 15% colony formation efficiency in
soft agarose (Fig. 4B). Since the A3 and A13 cells were
the only two lines that displayed zinc-inducible cyclin A protein
expression among 25 characterized G418-resistant clones(11) ,
assessment of phenotypic variation in cyclin A-mediated
anchorage-independent growth is limited. These results, however, are
consistent with previous studies indicating that a stable NRK cell line
overexpressing ectopic cyclin A is able to grow in suspension, whereas
the parental NRK fibroblasts are anchorage-dependent(8) .
Figure 4:
A, ectopic expression of cyclin A
is inducible by 50 µM zinc in non-adherent Rat 1a cell
lines A3 and A13, which were stably transfected with a zinc-dependent
human cyclin A expression plasmid. One day after plating in
zinc-supplemented medium, extracts from equal numbers (10)
of unattached cells were subjected to immunoblotting using a polyclonal
anti-cyclin A antibody (see Fig. 3A). B,
anchorage-independent growth of Rat1a cells induced to express cyclin A
ectopically. The figure shows Rat 1a cells and Rat 1a-InCycA A3 and A13
cells that were cultured in soft agar supplemented with no
Zn
(upperpanels) or 50 µM Zn
(lowerpanels). Whereas
Rat1a cells do not form anchorage-independent colonies, Rat1a-InCycA A3
and A13 cells in zinc form 10
and 1.5
10
colonies (diameter, >100 µm; averaged from
quadruplicates), respectively, per 10
cells
plated.
We
determined whether ectopic inducible expression of cyclin A may
increase endogenous Rat 1a myc expression, thereby
contributing indirectly to anchorage-independent growth. In
non-adherent Rat1a cells, induction of cyclin A expression did not
alter the low levels of endogenous rat myc mRNA (data not
shown). These results indicate that deregulated cyclin A expression
does not increase the endogenous myc mRNA level and is
sufficient to support adhesion-independent growth of Rat 1a cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.