RESPONSE OF THYROID FOLLICULAR CELLS TO PROTONS AND GAMMA IRRADIATION: The Role of Connexin32

LM Green 1,2,3,4, DK Murray1,2, DT Tran1, 3, SS Rightner1, S Todd1, B Bianski1 & GA Nelson1
1Radiobiology Program-Radiation Medicine, Loma Linda University, Loma Linda, CA; 2Molecular Immunology, JL Pettis Memorial Veterans Medical Center, Loma Linda, CA; Department of Microbiology & Molecular Genetics and School of Medicine, Loma Linda University
E-mail - lgreen@dominion.llumc.edu

INTRODUCTION

The "contact effect", wherein cells in an organized assembly are more resistant to the damaging effects of radiation than their monolayer counterparts, is a well-established finding.  Three possible, but not mutually exclusive, mechanisms for the contact effect have been proposed.  They involve gap junction intercellular communication, signal transduction/gene expression, and chromatin conformation.  In this regard, gap junction intercellular communication has been suggested to allow for the exchange of molecules essential to control of DNA repair and thus, enhance radiation resistance of cells growing in multicellular assemblies. This suggestion is based on gap junction "reciprocity", which describes the ability of cells to bidirectionally exchange information.  For example, healthy cells can provide necessary signals to rescue damaged cells, or conversely, damaged cells can dissipate or dilute toxic substances or damage signals by passing them to neighboring cells, thus increasing the recovery potential of the associated population.  Support for gap junctions contributing to the contact effect was obtained when it was demonstrated that certain cells electrically coupled via gap junction, when grown as spheroids, were relatively more radioresistant than uncoupled cells.  However, cells coupled in monolayers were not spared relative to single cells, suggesting that additional factors in three-dimensional cell structured microenvironments are contributing to these protective responses.

The definitive components of higher-order cellular assemblies responsible for the contact-effect have not been clearly defined.  Both adhesion molecules and gap junction proteins are implicated.  Together adhesion proteins and gap junctions provide the framework for normal cellular integration but their relative importance may be related to the specific properties of the cell types involved. Thus, gap junctions and adhesion molecules appear to play roles in regulating the responses of cells and tissues to irradiation according to specific programs.  Better understanding the pathways and rules governing these important cellular components will reveal potential sites of intervention.   It would be of great therapeutic benefit to be able to enhance the radiosensitivity of transformed cells and tumors, while sparing the surrounding normal cells/tissue.

Our previous studies have demonstrated that early passage (<15) follicular cultures of Fischer rat thyroid cells (FRTL-5) express functional connexin32 gap junctions.  Later passage (>15) cultures express a truncated non-functional form of the connexin32 protein and divide on average 30% faster than early passage cultures.  This model provided the opportunity to assess the role of connexin32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions.   

In this study we assess the contribution of functional Cx32 gap junctions to the radioresistance of thyroid follicular cultures exposed to gamma rays and proton beams delivered acutely over a range of physical doses.   By comparing cellular responses to qualitative differences in the patterns of radiation induced damage we attempted to reveal features of the underlying regulatory processes modulated by junctions and add to our understanding of how normal tissue reacts to therapeutic protons.

METHODS

All methods have been previously published (Green et al. 2001, Radiat Res 155:32-42; & Green et al. 2001 Mol Endocrinol In Press).  Briefly, FRTL-5 cells were purchased from the ATCC.  Cx32 gap junctions were down-regulated with 3mM heptanol added to cultures 2 hours prior to irradiation.  An El Dorado cobalt-60 unit supplied the g-irradiation (doses ranged from 0 Gy to 12 Gy) delivered at a rate of approximately 1.3 Gy/min.  Protons (250MeV) were delivered from the Loma Linda University Proton Treatment Facility synchrotron accelerator.  The proton dose range was 0 to 10Gy at a dose rate of approximately 0.7 Gy/min. The survival experiments were performed in 5-7 duplicate sets of FRTL-5 cells established in flasks.  Ninety-six hours post irradiation the flasks were fixed, stained with crystal violet and counted using an automated image processing system with trans-illumination.  A linear quadratic model was used to determine the a (initial slope, or shoulder of the curve) and b (terminal or descending slope) components of the survival curves.  The equation took the form of: surviving fraction (dose) =exp -a(D)-b(D)^2.

RESULTS

The survival curves generated following gamma irradiation revealed that early passage cultures had significantly lower a values (0.04 Gy-1) than later passage cultures (0.11 Gy-1)  (p<0.0001, n=10).   As an additional way to determine whether the presence or absence of Cx32 was contributing to the difference in survival, and whether the variation could, in part, be due to their growth rate differences, we treated the cultures with heptanol which reversibly down-regulates gap junctions without affecting their cell cycle.  The a-component of the survival curves obtained for early passage cultures treated with heptanol   (0.10  Gy-1 ) were nearly equivalent to the a values obtained for untreated late passage cultures  (0.11 Gy-1) (p>0.1,  n=8).   This data indicates that the presence of functional Cx32-type gap junctions contributes to the radiation resistance of the thyroid follicles in response to gamma irradiation.   Early and late passage cultures exposed to protons had survival curves and a values that were not significantly different when: the cells were expressing functional Cx32 (early passage cultures, a value 0.10 Gy-1); not expressing Cx32 (late passage cultures, a value 0.09 Gy-1); or were down-regulated with heptanol (early plus heptanol, a value 0.09 Gy-1; late plus heptanol, a value 0.12 Gy-1) (p>0.1).   Thus, for proton irradiation the presence or absence of Cx32-type gap junctional channels did not influence their radiosensitivity.

CONCLUSIONS

Collectively, the data supports the following conclusions. 1) The flatter shoulder (lower a value) of the gamma survival curves from the early passage cultures suggests greater repair efficiency and/or enhanced resistance to radiation induced damage, coincident with the expression of Cx32-type gap junctions. 2) The heptanol experiments indicate that protection provided by the gap junctions was only necessary during the first few hours post gamma irradiation. 3) In this model system the response of the irradiated follicular cultures was not dependent on their rate of cell division. 4) The lack of resistance and/or less efficient repair processes introduced by proton irradiation were independent of the contact-dependent intercellular communication status of the target cells. and 5) The fact that the b components of the survival curves were not significantly different (average 0.022+/- 0.003 Gy-2 , p> 0.1, n=20) for gamma rays or protons suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of both radiation quality and the presence of functional gap junctions. Thus, proton irradiated cells do not appear to be undergoing effective repair processes even at the lower doses implemented, implies that the effects of proton irradiation are overwhelming any sparing effects that Cx32 gap junctions/cellular integration could impart.

This work was supported in part by a NASA-Cooperative Research Agreement # NCC9-79