Radiation-induced differentiation of human skin fibroblasts: relationship with cell survival and collagen production

Abstract

The aim of this study was to determine whether significant inter-individual differences exist between skin fibroblast strains obtained from radiotherapy patients in both radiation-induced differentiation and collagen production in vitro, for use as potential parameters for a predictive assay for fibrosis following radiotherapy in patients. Morphological cell differentiation was determined 7 days after irradiation in seven early-passage primary human fibroblast cell strains and correlated with cell survival. Collagen production was measured in two cell strains by flow cytometry and incorporation of 3H-proline. There was a wide variation in the extent of radiation-induced differentiation for the seven cell strains, each showing a dose-related increase. The correlation between induced differentiation and cell survival was poor (r = 0.64) but statistically significant (p < 0.01). Collagen synthesis increased 7 days after irradiation for one cell strain (HF-48), as measured by incorporation of 3H-proline, but not in radiation sensitive AT-1 cells. The collagen I content of the two cell strains was assessed by flow cytometry but no significant differences were observed between the strains tested or with increasing dose. In conclusion, marked variations in radiation-induced fibroblast differentiation were observed between patients, this being an important criterion for a predictive assay.

Diversity of Fibroblasts – A Review on Implications for Skin Tissue Engineering

“…In contrast, the feeder layer system used to culture keratinocytes utilizes PMF, originally derived from the irradiated transformed mouse 3T3 cell line for coculture [Rheinwald and Green, 1975]. There is evidence that irradiation does not necessarily kill the feeder cells, but rather induces terminal differentiation with irreversible growth arrest [Rodemann and Bamberg, 1995], yet largely preserves physiologic function in producing growth factors and extracellular matrix proteins [Rodemann and Bamberg, 1995;Lara et al, 1996, Hakenjos et al, 2000von Pfeil et al, 2002]. Thus, the functional influence of the secreted cocktail of growth factors and cytokines by the various subpopulations of fibroblasts on keratinocyte proliferation remains unclear.…”

“…replicative progenitor fibroblasts (MF) Fibroblast Diversity Cells Tissues Organs 2008;187:165-176 167 and irreversible postmitotic fibrocytes (PMF). These two subsets have subsequently been classified by cytomorphological and biochemical characteristics [Bayreuther et al, 1988;Rodemann, 1989;Rodemann and Mueller, 1990;Rodemann and Bamberg, 1995;Lara et al, 1996;Rodemann et al, 1996;Burger et al, 1998;Herskind et al, 1998;Hakenjos et al, 2000]. Based on in vitro and ex vivo-in vitro experiments, it was shown that fibroblasts of human, rat and mouse skin, but also lung fibroblasts, differentiate in vivo and in vitro along a lineage of replicative MF cell types and nonreplicative, functional PMF of man, rat and mouse [Mollenhauer and Bayreuther, 1986;Bayreuther et al, 1988].…”

Fibroblast Differentiation in Subcutaneous Fibrosis after Postmastectomy Radiotherapy

“…Thus, 70 -100% of colony-forming progenitor fibroblasts inactivated by irradiation with 3.5 Gy could be accounted for by an increase in the number of postmitotic fibrocytes. This net change from colonies to single PMF cells demonstrates radiation-induced premature terminal differentiation in the cellular radiation response of fibroblasts as described by Rodemann et al, and others (15,16). In the previous studies, this was shown by a dose-dependent increase in the fraction of PMF cells scored in colonyforming cultures seeded at densities in the range 12 -50 cells/cm 2 .…”

Consequences of Radiotherapy for Breast Reconstruction

“…The increase in collagen production shows a dose response relationship in that the higher the radiation dose, the more collagen production is observed (Lara et al 1996). Ex-vivo studies of primary fibroblast cultures have shown that there is a wide inter-patient variation in the response of fibroblasts to irradiation; both in the intrinsic cellular radiosensitivity (cell killing) as in the degree of induction of terminal differentiation (Herskind et al 1998a;Herskind et al 2000;Johansen et al 1994;Lara et al 1996). This is also reflected by clinical observation: for a standard radiation dose there is a large variation in the degree of fibrosis development observed between different patients after radiation treatment for breast cancer, both after breast conserving treatment and after mastectomy (Bentzen et al 1989;Bentzen et al 1993;Borger et al 1994;Collette et al 2008) ( figure 10).…”

“…The increase in collagen production shows a dose response relationship in that the higher the radiation dose, the more collagen production is observed (Lara et al 1996). Ex-vivo studies of primary fibroblast cultures have shown that there is a wide inter-patient variation in the response of fibroblasts to irradiation; both in the intrinsic cellular radiosensitivity (cell killing) as in the degree of induction of terminal differentiation (Herskind et al 1998a;Herskind et al 2000;Johansen et al 1994;Lara et al 1996).…”