Cancer Research Landon Prizes for Basic and Translational Cancer Research AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 4843-4847, October 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dimitrijevic-Bussod, M.
Right arrow Articles by Gadbois, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dimitrijevic-Bussod, M.
Right arrow Articles by Gadbois, D. M.
[Cancer Research 59, 4843-4847, October 1, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Extracellular Matrix and Radiation G1 Cell Cycle Arrest in Human Fibroblasts1

Mira Dimitrijevic-Bussod, Viviana S. Balzaretti-Maggi and Donna M. Gadbois2

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is thought that sublethal doses of radiation cause cells to pause in either G1 or G2 phase, but that then cells with repaired DNA damage reenter the cell cycle. However, it has been observed that {gamma}-irradiation causes normal human fibroblasts to arrest indefinitely in G1 phase unless the irradiated cells are subcultured. This indicates that cell adhesion plays a role in maintaining the arrest. We now show that the type of extracellular matrix dramatically affects the percentage of cells that arrest in G1 phase. The prolonged radiation G1 arrest in human fibroblasts has been referred to as "senescence-like"; however, we find that smooth muscle {alpha}-actin is highly expressed in cells that arrest in G1 phase after irradiation. This indicates that the fibroblasts differentiate to myofibroblasts. Together, our results show that the length of radiation G1 arrest in human fibroblasts is affected by the type of extracellular matrix on which the cells are irradiated and that arrest results in myofibroblast differentiation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two temporally discrete cell cycle delays occur in cells in response to radiation: (a) one is late in G1 phase, before DNA replication; and (b) the other is in G2 phase, before mitosis. It is commonly proposed that these cell cycle delays provide cells with additional time to repair DNA damage and thereby promote genomic stability (1) . A cell must have an intact p53 pathway to delay in G1 phase after irradiation (2) . Irradiation of p53-positive cells results in increased p53 protein amounts, which subsequently cause an increase in p21Cip1 mRNA and protein. The p21Cip1 protein then mediates the G1 arrest by inhibiting CDK23 /cyclin E kinase activity, which retains the retinoblastoma protein in the hypophosphorylated form (3) . p53 is required to sustain a G2 arrest after high doses of radiation but not to initiate a G2 delay (4) . Rather, the predominate mechanism of G2 delay is by inhibiting cdc2/cyclin B kinase activity because of phosphorylation of tyrosine 15 on cdc2. This occurs through activation of wee1 kinase and inhibition of cdc25C phosphatase by chk1 kinase and 14-3-3 sequestering protein (reviewed in Ref. 5 ).

Until recently, it was thought that sublethal doses of radiation cause cells to pause in either G1 or G2 phase, and that then cells with repaired DNA damage reenter the cell cycle. This is largely true with the G2 delay. However, a variation of this theme has emerged when considering G1 delays in anchorage-dependent, primary human fibroblasts. It has now been observed that human fibroblasts that delay in G1 phase immediately after irradiation do not reenter the cell cycle (6 , 7) . Also, human fibroblasts, which are beyond the G1 checkpoint, delay in G2 phase but then divide and rearrest indefinitely in the next G1 phase. The prolonged G1 arrest occurs even at doses of 1 Gy, at which there is very little residual DNA damage. This arrest was called senescence-like because it was thought to be irreversible, because of morphological similarities to senescent cells, and because cellular p21Cip1 amounts remain elevated (6) . More recently, we have shown that the "permanent" radiation G1 arrest in human fibroblasts is reversible in many of the cells, if they are subcultured (8) . These results indicate that cell adhesion plays a role in maintaining the arrest.

Cell adhesion and the ECM are involved in signal transduction during a variety of cellular functions such as cell motility, morphogenesis, differentiation, and proliferation (reviewed in Refs. 9, 10, 11 ). G1 phase CDKs are regulated by cell adhesion in primary, anchorage-dependent fibroblasts (reviewed in Ref. 11 ). Mitogenic stimulation of suspended, quiescent fibroblasts causes G0 to G1 progression; however, the cells arrest in G1 phase because both CDK4/cyclin D and CDK2/cyclin E kinases remain inactive. Fibroblasts that are put into suspension in late G1 phase contain hyperphosphorylated retinoblastoma protein and are resistant to cell cycle inhibition (12) . These cells progress through the cell cycle and arrest in the next G1 phase after one cell division.

Radiation and suspension of anchorage-dependent fibroblasts cause G1 arrest because of the inhibition of G1 CDKs. Indeed, our data indicate that cell adhesion affects the ability of fibroblasts to undergo and maintain the radiation G1 arrest (8) . A major mode of cell adhesion is via the interaction of integrins with ECM ligands, e.g., fibronectins, collagens, and laminins. Integrins form a family of heterodimeric cell surface receptors composed of {alpha}/ß subunits (reviewed in Ref. 13 ). The identity of the {alpha}/ß subunits determines the ECM ligand specificity. Particular integrin/ECM interactions lead to distinct cellular responses, such as cell proliferation and differentiation (Refs. 14 and 15 ; reviewed in Ref. 16 ).

Our studies show that disrupting cell adhesion affects the longevity of the radiation G1 arrest in human fibroblasts; hence, we hypothesize that changing the type of adhesion interactions will alter or alleviate the arrest. To address this proposal, we measured cell cycle delays after {gamma}-irradiation of human fibroblasts on the substrates polystyrene, Mylar, collagen IV, fibronectin, and laminin. We found dramatic differences in the percentage of cells that undergo a sustained G1 arrest after irradiation on the different substrates. Furthermore, substrates that promote a prolonged radiation G1 arrest also cause an increase in SM {alpha}-actin expression in the arrested cells, which is characteristic of the myofibroblast phenotype (17) . These results indicate that specific types of adhesion interactions contribute to the maintenance of the G1 arrest observed after irradiation of human fibroblasts and that the arrest culminates in the differentiation of fibroblasts to myofibroblasts, rather than senescence.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Irradiation.
HSF cells were derived from neonatal foreskin and cultured at 37°C under 5% CO2:air in {alpha}-MEM supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin. Mycoplasma-free cells from passage 8–12 were used. Polystyrene culture flasks were purchased from Costar (Cambridge, MA). Mylar culture dishes were custom-fabricated as described in previous studies (7) . Human collagen IV, murine laminin, and human fibronectin culture flasks were obtained from BioCoat Cellware (Becton Dickinson Labware, Bedford, MA).

For {gamma}-ray exposures, cells were seeded at an initial density of 5.3–6.7 x 103 cells/cm2 24 h before irradiation. A Mark I model 68A high-dose rate 137Cs source chamber irradiator (J. L. Shepherd and Associates) was used to irradiate cells with 2 Gy of {gamma}-rays at a dose rate of 2.1 Gy/min at room temperature. BrdUrd (Sigma, St. Louis, MO) was added directly to the culture medium at a final concentration of 10 µM at the indicated times after irradiation.

Flow Cytometric Analyses.
Flow cytometric analysis was performed as described previously (7) . Cells were harvested using 0.25% trypsin/1 mM EDTA, fixed in 70% ethanol for at least 1 h, and then stained overnight at 4°C in PBS containing 0.5 µg/ml HO (Calbiochem, San Diego, CA), 5 µg/ml MI (Pfizer Corp., Groton, CT), and 5 mM MgCl2 at a cell concentration of 7.5 x 105 cells/ml. A two-laser flow cytometer was used for cell cycle analysis, with one laser operating in the UV region (333.6–363.8 nm), and one tuned to 547.9 nm. The lasers were separated by 250 µm to provide sequential excitation and analysis of each fluorochrome. HO emissions were measured over a 400–495-nm range, and MI fluorescence was measured above 495 nm. The electronic gains were adjusted so that the BrdUrd-negative G1 peaks of the HO and MI DNA content histograms were initially in the same channel number, and the fluorescence signals were then compared electronically on a cell-cell basis. Such comparisons reflect the quenching of the HO fluorescence by BrdUrd, which is proportional to cellular BrdUrd content in cells that had synthesized DNA during the labeling period.

Western Analysis.
Cells were harvested using 0.25% trypsin/1 mM EDTA and washed with PBS, and cell pellets were stored at -70°C. Protein lysates were prepared as described previously (7) . Protein concentration was determined using the DC protein assay (Bio-Rad, Hercules, CA), and equal amounts of protein (8–10 µg) were resolved on 10% or 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Equal loading and transfer were ensured by Ponceau S staining of the membranes. Specific proteins were detected using a mouse monoclonal antibody to p53 (DO-1); rabbit polyclonal antibodies to p21Cip1 (C-19), CDK2 (M2), or CDK4 (C-22; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); or a mouse monoclonal antibody to SM {alpha}-actin (Klon 1A4; DAKO, Carpinteria, CA). Horseradish peroxidase-linked secondary antibodies (Amersham) and Renaissance chemiluminescence reagents (DuPont New England Nuclear, Boston, MA) were used to visualize proteins.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Cycle Arrest on Mylar or Polystyrene.
Several studies show that ionizing radiation causes normal human fibroblasts to accumulate in the G1 phase of the cell cycle in an apparently permanent arrest (6 , 7) . However, we have shown that the arrest is reversible in many cells if cell adhesion interactions are interrupted by subculturing the radiation-arrested cells (8) . If cell adhesion influences radiation arrest in human fibroblasts, then particular integrin interactions with certain ECM ligands might lead to varied cell cycle responses to radiation. In an initial attempt to test this hypothesis, we compared the radiation arrest we have observed on tissue culture polystyrene to radiation arrest in fibroblasts grown on Mylar. We chose to test Mylar because it is a commonly used culture substrate when irradiating cells with {alpha}-particles. Also, it has been shown that the surface chemistry of the plastic polymer dictates the amounts and conformation of adsorbed ECM molecules (18, 19, 20, 21) .

HSF cells were irradiated with 2 Gy of {gamma}-rays on either polystyrene or Mylar, and cells were labeled with BrdUrd for 24 h before the given harvest times (Fig. 1)Citation . Even at 24 h after irradiation, there is an increase in BrdUrd-labeled/proliferating cells in Mylar cultures (78%) compared to polystyrene (65%) cultures. However, the effect of substrate on G1 arrest is dramatically illustrated in the 48 h time point. A total of 74% of HSFs in Mylar cultures but only 28% of cells in polystyrene cultures become BrdUrd-labeled 24–48 h after irradiation. Also, many proliferating cells in the Mylar cultures are in the S phase and G2 phase of the second cell cycle (Fig. 1Citation , arrows, 48 h), whereas cells that divide in the polystyrene cultures remain in G1 phase.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 1. Proliferation of HSF cells after irradiation on polystyrene or Mylar. The percentages shown for each time point are the percentages of cells that became labeled with BrdUrd during the 24-h period before the given harvest time. These percentages correspond to the large gated areas. A differential DNA dye procedure was used to determine the uptake of BrdUrd into the DNA of proliferating cells. Cells are labeled with BrdUrd and then stained with both MI (x-axis) and HO (y-axis). MI fluorescence is affected negligibly by BrdUrd and therefore represents the cell cycle phase of a particular cell. HO fluorescence is quenched by DNA-bound BrdUrd; therefore, cells that proliferate during the BrdUrd labeling period have a decrease in HO fluorescence. In the polystyrene 24 h time point, cells in various phases of the cell cycle are gated for the readers convenience. UG1, unlabeled G1 cells; LS1, BrdUrd-labeled first cycle S phase; UG2, unlabeled G2, LG1, labeled G1; LS2, labeled second cycle S phase; LG2, labeled G2. All flow cytometry experiments in this study were performed at least three times, and a representative experiment is shown.

 
At 48–72 h after irradiation, 81% of the cells on Mylar are BrdUrd-labeled compared to 34% of cells in the polystyrene cultures. Mylar cultures contain many cells that are present in the second S phase and G2 phase, whereas such cells are absent in the polystyrene cultures (Fig. 1Citation , arrows, 72 h). In control experiments, HSF cells that are cultured on polystyrene or Mylar grow at similar rates, so the difference in proliferation after irradiation is not due to a general difference in cell kinetics or cell cycle distribution at the time of irradiation.

Human fibroblasts that undergo a late G1 arrest after {gamma}-irradiation have a senescence-like phenotype in that the cells become very large and flat on the culture dish. We observed that cells irradiated on polystyrene are very large and flat after irradiation (data not shown). In contrast, cells that were irradiated on Mylar retained a smaller, more normal appearance, which correlates with our observation that HSF cells that are irradiated on Mylar do not remain arrested in G1. Together, the data described in this section indicate that fewer fibroblasts that are irradiated on Mylar arrest permanently in G1, and they also do not take on a senescence-like phenotype. Although the expression pattern of adhesion molecules on polystyrene and Mylar is not known, these data provide proof-of-principle support for our hypothesis that changing the type of adhesion interactions alters the percentage of HSF cells that arrest in G1 after irradiation.

Radiation Arrest on Collagen IV, Fibronectin, or Laminin.
We next determined the effect of naturally occurring ECM molecules on radiation G1 arrest of HSF cells. HSF cells were irradiated with 2 Gy of {gamma}-rays on polystyrene, collagen IV, fibronectin, or laminin, and proliferating cells were labeled with BrdUrd for 24 h before the given harvest times. The total percentage of BrdUrd-labeled cells (proliferating cells) was determined using flow cytometry as described in Fig. 1Citation , and a graphic representation of this data is shown in Fig. 2Citation . The percentage of BrdUrd-labeled cells ranges from about 58% on polystyrene to about 70% on laminin during the first 24 h after irradiation. The difference in proliferation is much more dramatic at the 48 h time point, with only about 22% BrdUrd-labeled cells on polystyrene, 40–43% BrdUrd-labeled cells on fibronectin or collagen IV, and over 60% BrdUrd-labeled cells on laminin. At 72 h, there are only about 20% BrdUrd-labeled cells on polystyrene, 40% cycling cells on collagen IV or laminin, or about 60% BrdUrd-labeled cells on laminin. In Fig. 2Citation , the percentage of cycling, irradiated cells on fibronectin and collagen is nearly identical. However, we sometimes find that the percentage of cycling cells on fibronectin is 5–10% higher than on collagen IV. Therefore, the trend that we observe for the percentage of cycling, irradiated HSF cells on different substrata is as follows: Mylar > laminin > fibronectin >= collagen IV > polystyrene.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Proliferation of HSF cells after irradiation on laminin, fibronectin, collagen IV, or polystyrene. Flow cytometric analysis of irradiated HSF cells was performed as described in Fig. 1Citation . The proliferating (BrdUrd-labeled) cells were gated, and the percentage of proliferating cells is graphed as a function of time after irradiation.

 
p53, p21Cip1, CDK2, and CDK4 Expression.
G1 phase radiation arrest is initiated by an increase in p53 protein amounts, followed by the induction of p21Cip1 mRNA and protein (2 , 3) . This results in an inhibition of G1 phase CDKs and G1 phase arrest (3) . The percentage of HSF cells that remain arrested in G1 phase after irradiation differs, depending on the ECM substrate. Therefore, we determined whether amounts of p53, p21Cip1, CDK2, and CDK4 correlate with the percentage of cells that arrest in G1 after irradiation.

Our data indicate that the amount of induction of p53 is not a controlling factor in the length of the substrate-dependent radiation arrest. In all cases, p53 is induced at early times after irradiation and remains elevated at late times after irradiation when there are large differences in the percentage of proliferating cells (Fig. 3)Citation .



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Western blot analysis of cell cycle protein expression in HSF cells irradiated on Mylar, laminin, fibronectin, collagen IV, and polystyrene.

 
There are subtle but reproducible differences in the expression of p21Cip1, CDK2, and CDK4 (Fig. 3)Citation . Irradiation of HSF cells on polystyrene, collagen IV, or fibronectin (where a high percentage of cells arrest) causes an increase in p21Cip1 by 4 h after irradiation, which then decreases to a constant but elevated level by 48 h after irradiation. Many cells escape the late arrest when irradiated on laminin, and p21Cip1 is induced at a later time compared to polystyrene, collagen IV, and fibronectin. On Mylar (where few cells arrest), p21Cip1 is only modestly induced but remains slightly elevated throughout the experiment.

The expression patterns of CDK2 and CDK4 are more complex. For example, the amount of CDK2 decreases dramatically in polystyrene cultures, whereas there is only a modest decrease in CDK2 amounts of HSF cells irradiated on Mylar, where many cells are proliferating. Lower amounts of p21Cip1 and higher amounts of CDK2 in Mylar cultures might correlate with higher CDK2 activity and an increase in proliferation. However, the amounts of CDK2 also decrease in laminin cultures, and many of the cells escape the late arrest. CDK4 amounts increase at late times after irradiation on laminin and Mylar. Increased proliferation after irradiation on these substrates might be supported by increased CDK4 activity or a titration of p21Cip1 by increased CDK4, which could allow lower amounts of CDK2 to remain active.

SM {alpha}-Actin Expression during Radiation Arrest.
Radiation causes the differentiation of fibroblasts to myofibroblasts (22 , 23) . A molecular marker characteristic of myofibroblasts is SM {alpha}-actin (17) . Therefore, we determined whether radiation cell cycle arrest of HSF cells on different substrates is accompanied by an increase in SM {alpha}-actin expression. As seen in Fig. 4Citation , SM {alpha}-actin is induced during the late arrest on polystyrene, collagen IV, and fibronectin, but not when HSF cells remain proliferating on laminin or Mylar. These data show that fibroblasts take on a differentiated myofibroblast phenotype after irradiation that is ECM dependent.



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 4. Western blot analysis of SM {alpha}-actin expression in HSF cells irradiated on Mylar, laminin, fibronectin, collagen IV, and polystyrene.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The dogma that radiation cell cycle delays occur primarily to repair DNA damage is well accepted but has not been rigorously proven. There is no doubt that DNA is the target of radiation in terms of cellular survival (see Ref. 24 ). It is also clear that DNA damage is a signal for increased p53 expression, which then results in cell cycle delay (25) . The purpose of cell cycle delay is less obvious. One of the few studies that directly tests the idea that cell cycle delay results in increased repair of DNA damage shows that there is increased survival if cells are held in confluence-induced G0 phase for several hours after irradiation (26) . This indicates that, indeed, cell cycle delay after irradiation allows time to repair DNA damage. However, this is a very simplistic viewpoint that does not consider the potential function of individual cell types during radiation stress response in a tissue environment. For example, epithelial cells, which are a primary target of carcinogenesis, may apoptose after irradiation to subtract damaged cells from the reproductive population. Fibroblasts provide signals to epithelial cells during growth, development, differentiation, and apoptosis (reviewed in Refs. 27 and 28 ). Therefore, radiation may cause fibroblasts to undergo cell cycle arrest and then differentiation to provide specific stress signals to neighboring cells. In this study, we demonstrate that fibroblasts arrest in G1 phase indefinitely and then differentiate to myofibroblasts, depending on the type of ECM environment. By inducing differentiation, the ECM may influence the type of signals that irradiated fibroblasts provide to surrounding cells. These results indicate that the interpretation of radiation cell cycle responses must be expanded to include the extracellular environment, in particular, the ECM.

We demonstrate that the ECM affects the percentage of HSF cells that remain arrested in G1 phase after irradiation. Dramatic differences in proliferation after irradiation on polystyrene or Mylar are observed. The significance of this is not immediately apparent because plastic is not a biological substrate. However, ECM molecules in the serum or that are synthesized by the cells rapidly coat the plastic. It is this layer of conditioning protein that controls cell adhesion and subsequent cellular processes. The amount and orientation of protein adsorption to a substrate is controlled by the chemistry of the polymer surface (Ref. 18 and references therein). For example, the amount of laminin 5 that coats polystyrene depends on the type of functional groups that are created on the plastic during manufacturer’s plasma treatment of culture dishes (19) . Surface chemistry also affects the orientation of bound laminin 5, resulting in differences in the availability of integrin-binding sites (28) . Substrata chemistry also determines the conformation of bound fibronectin and, in turn, RGDS-dependent adhesion of fibroblasts and differentiation of neuronal cells (20 , 21) . The functional groups in plasma-treated polystyrene and Mylar are vastly different. Perhaps this influences the composition and/or orientation of adsorbed ECM and results in an altered cell cycle response to radiation. This is supported by our observation that radiation causes a different percentage of HSF cells to arrest on the naturally occurring ECM substrates, collagen IV, fibronectin, and laminin.

It is interesting that irradiated fibroblasts do not arrest well or differentiate on laminin because laminin is not an ECM protein that is commonly thought to be associated with the stroma. Rather, laminin is associated with the basement membrane and interacts more with epithelial cells. However, it has been found that transformed fibroblasts exhibit increased expression of integrin {alpha}6, a component of the laminin receptor (29) . Also, the expression of the fibronectin receptor {alpha}5ß1 is lower in transformed fibroblasts than in normal fibroblasts (30) . Another indication that integrin expression is involved in tumorigenesis comes from studies that showed that treatment of malignant breast cells with an inhibitory antibody to integrin ß1 results in differentiation/reversion of the malignant phenotype (31) . Perhaps the irregular expression pattern of integrins in transformed fibroblasts or an aberrant ECM environment around nontransformed fibroblasts would result in an abnormal cell cycle response to radiation or other stress insults.

We demonstrate that the prolonged G1 arrest results in SM {alpha}-actin expression, a hallmark of the myofibroblast phenotype. Several studies have reported that radiation causes fibroblast differentiation (22 , 23) , and ECM control of differentiation is well documented (reviewed in Ref. 16 ). However, to our knowledge, this is the first report of an ECM dependence of radiation-induced differentiation of fibroblasts. The SM {alpha}-actin promoter is p53 responsive (32) . It is unlikely that p53 induction is solely responsible for increased SM {alpha}-actin expression because p53 amounts increase within the first few hours after irradiation, whereas increases in SM {alpha}-actin begin much later. Also, p53 amounts remain elevated on all substrates, and SM {alpha}-actin expression increases only on substrates where there is a late G1 arrest. Therefore, some other molecular change, in addition to p53 expression, probably occurs in order for SM {alpha}-actin to be induced.

Perhaps the production of an extracellular molecule is involved in myofibroblast differentiation because radiation causes both an increase in TGF-ß1 and fibroblast differentiation (22 , 23 , 33 , 34) . These two events might be inter-related because direct treatment of human and rat fibroblast cultures with activated TGF-ß1 causes differentiation to myofibroblasts (35, 36, 37) . In fact, TGF-ß1 induction of specific ECM proteins might evoke myofibroblast differentiation. TGF-ß1 expression leads to a large increase in the amounts of the ED-A splice form of fibronectin in cultured fibroblasts (38) . The increase in ED-A fibronectin expression is followed by SM {alpha}-actin expression (39) . Plating fibroblasts in the presence of neutralizing antibodies to ED-A fibronectin blocks the induction of SM {alpha}-actin by TGF-ß1. It has been postulated that TGF-ß1 induction in fibroblasts causes a cell cycle arrest through the inhibition of G1 phase CDKs, which then predisposes the cells to differentiation (36) . The basal amounts of TGF-ß1 produced by myofibroblasts are 10–15 times higher than those produced by fibroblasts; however, radiation induces TGF-ß1 only in fibroblasts (36) . Thus, it was further suggested that the high basal amounts of TGF-ß1 in myofibroblasts maintain the differentiated phenotype. This scenario would agree with our data that show that cell cycle arrest occurs early after irradiation, whereas differentiation is a later event.

The function of myofibroblast differentiation is not known, but it may be involved in radiation fibrosis, a dose-limiting and debilitating side effect of radiation cancer therapy in which fibroblasts deposit copious amounts of collagen and other matrix proteins in the irradiated tissue. This results in the progressive formation of aberrant ECM and localized regions of fibrosis. It is thought that myofibroblasts support ECM remodeling in fibrotic tissue (see Ref. 36 ) and that changes in the differentiation pattern of fibroblasts reflect the risk of patients to develop fibrosis (36 , 40) . Radiation fibrosis is often compared to wound healing. In both, there is a remodeling of the ECM, an increase in TGF-ß1, and the differentiation of fibroblasts to myofibroblasts. The difference is that these events are reversed when the wound heals but are permanent in fibrotic tissue (41) .

Another possible function for myofibroblasts is in cell-cell signalling, as suggested in prostate tissue (42) . In the prostate gland, the epithelial cell mass is organized into distinct regions of viable or apoptotic epithelial cells. The surrounding stroma is also spatially organized: fibroblasts neighbor the regions of viable epithelial cells; and myofibroblasts neighbor areas of apoptotic epithelial cells. Androgen ablation causes an increase in TGF-ß1 and mass apoptosis of epithelial cells throughout the prostate gland. Concurrently, the number of fibroblasts in the stroma decreases, and myofibroblasts surround the epithelial mass. The accumulation of myofibroblasts is thought to be caused by the differentiation of preexisting fibroblasts, and it is proposed that the myofibroblasts are responsible for producing TGF-ß1 that leads to epithelial cell apoptosis. This is intriguing because human lung and prostate fibroblasts undergo a late radiation G1 arrest along with an induction of p53 and p21Cip1 proteins. However, cultured bronchial and prostate epithelial cells do not arrest and do not display an increased expression of p53 and p21Cip1 proteins (43 , 44) . Perhaps in vivo or in fibroblast-epithelial cell coculture, TGF-ß1 produced by myofibroblasts would cause cell cycle arrest or apoptosis of epithelial cells after irradiation.

The p53 control of radiation arrest has been well studied. However, there is a huge gap in our knowledge concerning the effects of the extracellular environment on radiation arrest. Also, there are several demonstrations that radiation causes fibroblast differentiation, but little is known about the significance of myofibroblast function or how the ECM affects myofibroblast differentiation. Obviously, the extracellular environment is of extreme importance because it more closely approaches the in vivo situation. Perhaps the phenomenon of permanent arrest of irradiated fibroblasts observed in vitro reflects the differentiation of fibroblasts in fibrosis and provides an opportunity to investigate whether cellular interactions with the ECM play an active role in this process.


   ACKNOWLEDGMENTS
 
We thank Robb Habbersett for expertise in flow cytometry and Jim Freyer and Karen LaRue for constructive criticism of the manuscript and experimental design.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Laboratory Directed Research Program of Los Alamos National Laboratory (Project Number 97021) and the National Flow Cytometry Resource (NIH, Contract Grant Number RR01315). Back

2 To whom requests for reprints should be addressed, at Life Sciences Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 667-2698; Fax: (505) 665-6894; E-mail: gadbois{at}telomere.lanl.gov Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; SM, smooth muscle; ECM, extracellular matrix; HO, Hoechst 33342; BrdUrd, bromodeoxyuridine; HSF, human diploid fibroblast strain 55; MI, mithramycin; TGF, transforming growth factor. Back

Received 5/26/99. Accepted 8/ 5/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lane D. P. p53, guardian of the genome. Nature (Lond.), 358: 15-16, 1992.[Medline]
  2. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wildtype p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA, 89: 7491-7495, 1992.[Abstract/Free Full Text]
  3. Dulic V., Kaufmann W. K., Wilson S. J., Tlsty T. D., Lees E., Harper J. W., Elledge S. J., Reed S. I. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell, 76: 1013-1023, 1994.[Medline]
  4. Bunz F., Dutriaux A., Lengauer C., Waldman T., Zhou S., Brown J. P., Sedivy J. M., Kinzler K. W., Vogelstein B. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science (Washington DC), 282: 1497-1501, 1998.[Abstract/Free Full Text]
  5. Weinert T. A DNA damage checkpoint meets the cell cycle engine. Science (Washington, DC), 277: 1450-1451, 1997.[Free Full Text]
  6. Di Leonardo A., Linke S. P., Clarkin K., Wahl G. M. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev., 8: 2540-2551, 1994.[Abstract/Free Full Text]
  7. Gadbois D. M., Crissman H. A., Nastasi A., Habbersett R., Wang S., Chen D., Lehnert B. E. Alterations in the progression of cells through the cell cycle after exposure to {alpha} particles or {gamma} rays. Radiat. Res., 146: 414-424, 1996.[Medline]
  8. Gadbois D. M., Bradbury E. M., Lehnert B. E. Control of radiation-induced G1 arrest by cell-substratum interactions. Cancer Res., 57: 1151-1156, 1997.[Abstract/Free Full Text]
  9. Sastry S. K., Horwitz A. F. Adhesion-growth factor interactions during differentiation: an integrated biological response. Dev. Biol., 180: 455-467, 1996.[Medline]
  10. Gumbiner B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell, 84: 345-347, 1996.[Medline]
  11. Assoian R. K., Zhu X. Cell anchorage and the cytoskeleton as partners in growth factor dependent cell cycle progression. Curr. Opin. Cell Biol., 9: 93-98, 1997.[Medline]
  12. Guadagno T. M., Assoian R. K. G1/S control of anchorage-independent growth in the fibroblast cell cycle. J. Cell Biol., 115: 1419-1425, 1991.[Abstract/Free Full Text]
  13. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69: 11-25, 1992.[Medline]
  14. Belkin A. M., Retta S. F. ß1D integrin inhibits cell cycle progression in normal myoblasts and fibroblasts. J. Biol. Chem., 273: 15234-15240, 1998.[Abstract/Free Full Text]
  15. Sastry S. K., Lakonishok M., Thomas D. A., Muschler J., Horwitz A. F. Integrin {alpha} subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J. Cell Biol., 133: 169-184, 1996.[Abstract/Free Full Text]
  16. Lin C. Q., Bissell M. J. Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J., 7: 737-743, 1993.[Abstract]
  17. Skalli O., Schurch W., Seemayer T., Lagace R., Montadon D., Pittet B., Gabbiani G. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab. Investig., 60: 275-285, 1989.[Medline]
  18. Slack S. M., Horbett T. A. Changes in the strength of fibrinogen attachment to solid surfaces: an explanation of the influence of surface chemistry on the vroman effect. J. Colloid Interface Sci., 133: 148-165, 1989.
  19. Fitchmun M. I., Falk-Marzillier J., Marshall E., Cruz G., Jones J. C. R., Quaranta V. Mode of adsorption and orientation of an extracellular matrix protein affect its cell-adhesion-promoting activity. Anal. Biochem., 265: 1-7, 1998.[Medline]
  20. Lewandowska K., Pergament B., Sukenik C. N., Culp L. A. Cell-type-specific adhesion mechanisms mediated by fibronectin adsorbed to chemically derivatized substrata. J. Biomed. Mater. Res., 26: 1343-1363, 1992.[Medline]
  21. Cheng S-S., Chittur K. K., Sukenik C. N., Culp L. A., Lewendowska K. J. The conformation of fibronectin on self-assembled monolayers with different surface composition: an FTIR/ATR study. J. Colloid Interface Sci., 162: 135-143, 1994.
  22. Rodemann H. P., Peterson H. P., Schwenke K., Von Wangenheim K. H. Terminal differentiation of human fibroblasts is induced by radiation. Scanning Microsc., 5: 1135-1143, 1992.
  23. Lara P. C., Russell N. S., Smolders I. J. H., Bartelink H., Begg A. C., Coco-Martin J. M. Radiation-induced differentiation of human skin fibroblasts: relationship with cell survival and collagen production. Int. J. Radiat. Biol., 70: 683-692, 1996.[Medline]
  24. Hall E. J. Ryan J. D. Cox K. Papdopoulos D. eds. . Radiobiology for the Radiobiologist, : 39-40, J. B. Lippincott Philadelphia 1994.
  25. Nelson W. G., Kastan M. B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol., 14: 1815-1823, 1994.[Abstract/Free Full Text]
  26. Little J. B., Nagasawa H. Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts. Radiat. Res., 101: 81-93, 1985.[Medline]
  27. Cunha G. R., Donjacour A. A., Cooke P. S., Mee S., Bigsby R. M., Higgins S. J., Sugimura Y. The endocrinology and developmental biology of the prostate. Endocr. Rev., 8: 338-363, 1987.[Medline]
  28. Ronnov-Jessen L., Peterson O. W., Bissell M. J. Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. Physiol. Rev., 76: 69-125, 1996.[Abstract/Free Full Text]
  29. Lin C-S., Zhang K., Kraemer R. {alpha}6 integrin is up-regulated in step increments accompanying neoplastic transformation and tumorigenic conversion of human fibroblasts. Cancer Res., 53: 2950-2953, 1993.[Abstract/Free Full Text]
  30. Plantefaber L. C., Hynes R. O. Changes in integrin receptors on oncogenically transformed cells. Cell, 56: 281-290, 1989.[Medline]
  31. Weaver V. M., Peterson O. W., Wang F., Larabell C. A., Briand P., Damsky C., Bissell M. J. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol., 137: 231-245, 1997.[Abstract/Free Full Text]
  32. Comer K. A., Dennis P. A., Armstrong L., Catino J. J., Kastan M. B., Kumar C. C. Human smooth muscle {alpha}-actin is a transcriptional target of the p53 tumor suppressor protein. Oncogene, 16: 1299-1308, 1998.[Medline]
  33. Barcellos-Hoff M. H. Radiation-induced transforming growth factor ß and subsequent extracellular matrix reorganization in murine mammary gland. Cancer Res., 53: 3880-3886, 1993.[Abstract/Free Full Text]
  34. Finkelstein J. N., Johnston C. J., Baggs R., Rubin P. Early alterations in extracellular matrix and transforming growth factor ß gene expression in mouse lung indicative of late radiation fibrosis. Int. J. Radiat. Oncol. Biol. Phys., 28: 621-631, 1994.[Medline]
  35. Desmouliere A., Geinoz A., Gabbiani F., Gabbiani G. Transforming growth factor-ß1 induces {alpha}-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol., 122: 103-111, 1993.[Abstract/Free Full Text]
  36. Burger A., Loffler H., Bamberg M., Rodemann H. P. Molecular and cellular basis of radiation fibrosis. Int. J. Radiat. Biol., 73: 401-408, 1998.[Medline]
  37. Peehl D. M., Sellers R. G. Induction of smooth muscle cell phenotype in cultured human prostatic stromal cells. Exp. Cell Res., 123: 208-215, 1997.
  38. Balza E., Borsi L., Allemanni G., Zardi L. Transforming growth factor ß regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS Lett., 228: 42-44, 1988.[Medline]
  39. Serini G., Bochaton-Piallat M-L., Ropraz P., Geinoz A., Borsi L., Zardi L., Gabbiani G. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-ß1. J. Cell Biol., 142: 873-881, 1998.[Abstract/Free Full Text]
  40. Herskind C., Bentzen S. M., Overgaard J., Overgaard M., Bamberg M., Rodermann H. P. Differentiation state of skin fibroblast-cultures versus risk of subcutaneous fibrosis after radiotherapy. Radiother. Oncol., 47: 263-269, 1998.[Medline]
  41. Darby I., Skalli O., Gabbiani G. {alpha}-Smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab. Investig., 63: 21-29, 1990.[Medline]
  42. Lee C. Role of androgen in prostate growth and regression: stromal-epithelial interaction. Prostate Suppl., 6: 52-56, 1996.[Medline]
  43. Gadbois D. M., Lehnert B. E. Cell cycle response to DNA damage differs in bronchial epithelial cells and lung fibroblasts. Cancer Res., 57: 3174-3179, 1997.[Abstract/Free Full Text]
  44. Girinski T., Koumenis C., Graeber T. G., Peehl D. M., Giaccia A. J. Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents. Cancer Res., 55: 3726-3731, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
W. Huang, R. Chiquet-Ehrismann, J. V. Moyano, A. Garcia-Pardo, and G. Orend
Interference of Tenascin-C with Syndecan-4 Binding to Fibronectin Blocks Cell Adhesion and Stimulates Tumor Cell Proliferation
Cancer Res., December 1, 2001; 61(23): 8586 - 8594.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
J. E. Feugate, Q. Li, L. Wong, and M. Martins-Green
The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure
J. Cell Biol., January 7, 2002; 156(1): 161 - 172.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dimitrijevic-Bussod, M.
Right arrow Articles by Gadbois, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dimitrijevic-Bussod, M.
Right arrow Articles by Gadbois, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online