©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclin A Links c-Myc to Adhesion-independent Cell Proliferation (*)

John F. Barrett , Brian C. Lewis (§) , Arthur T. Hoang , Rene J. Alvarez , Jr. , Chi V. Dang (¶)

From the (1)Division of Hematology, Department of Medicine and the Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

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).

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.


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).

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) .


RESULTS AND DISCUSSION

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).


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) .

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 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.

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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA57341 (to C. V. D.) and by a grant from the Wilbur-Rogers Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Howard Hughes Predoctoral Fellowship, Program in Human Genetics and Molecular Biology.

Leukemia Society of America Scholar. To whom correspondence should be addressed: Ross Research Bldg., Rm. 1025, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-2773; Fax: 410-955-0185; E-mail: cvdang@welchlink.welch.jhu.edu.

The abbreviation used is: NRK, normal rat kidney.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.