Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor

Purpose: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radioresponsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro). Methods and Materials: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy. DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis. Repair kinetics were studied at 37 degrees C for 4 h after irradiation, Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry. For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment. Results: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells, Compared to repair rates in in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity. Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor. A lower plating efficiency and differences in ploidy (as revealed by how cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics. Conclusions: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells

DEGRANULATION OF RAT SALIVARY-GLANDS FOLLOWING TREATMENT WITH RECEPTOR-SELECTIVE AGONISTS

1. The aim of this study was to find a drug that induces an almost complete degranulation of secretory cells in rat parotid and submandibular glands. 2. Phenylephrine (alpha-adrenergic), isoproterenol (beta-adrenergic) and mecholine (muscarinic cholinergic) were tested. Time and degree of maximal depletion of acinar and granular convoluted tubule cells were determined morphologically. 3. Following phenylephrine-injection (5 mg/kg or 10.2 mg/kg, i.p.), no effect on the acinar granulation level was observed in either of the glands, while about 50-60% granular convoluted tubules were degranulated for at least 120-180 min postinjection. 4. With isoproterenol (5, 10, 40, 70 or 100 mg/kg, i.p.), degranulation of 100% of the acinar cells in the parotid and 80% of the acinar cells in the submandibular gland was observed 90 min post-injection. Granular convoluted tubule cells did not respond to this beta-adrenergic drug. 5. Mecholine (3.75 or 7.5 mg/kg, i.p.) induced mainly degranulation of granular convoluted tubule cells (about 50% after 120 min). Numbers of granulated acinar cells decreased only slightly in both glands (about 10%, 90-120 min). 6. From this study it appears that with a relatively low dosage (5 mg/kg, i.p.) of isoproterenol, a high level of degranulation can be induced in acinar cells of rat parotid and submandibular glands without toxic side effects. Concerning granular convoluted tubules, only moderate degranulation was observed with phenylephrine and mecholine, respectively

THE ROLE OF SECRETORY GRANULES IN RADIATION-INDUCED DYSFUNCTION OF RAT SALIVARY-GLANDS

To investigate the possible role of secretory granules in radiation-induced salivary gland dysfunction, rats were pretreated with isoproterenol (5 mg/kg intraperitoneally) to degranulate salivary gland acini, At maximal depletion, salivary glands were locally irradiated with a single dose of 15 Gy of X rays. Parotid and submandibular/sublingual saliva samples were collected before and 1-10 days after irradiation, The lag phase, flow rate, concentrations of potassium and sodium, and amylase secretion were determined. Sham-treated, isoproterenol-treated and irradiated animals provided reference data. In the parotid gland, but not in the submandibular gland, protection against radiation-induced changes in flow rate and composition of saliva occurred after pretreatment with isoproterenol. Combining morphological data from a previous study with data from the current study, it is suggested that improvement of parotid gland function is attributed predominantly to a proliferative stimulus on acinar cells by isoproterenol and not to its degranulation effect, After pretreatment with isoproterenol, an earlier expression of radiation-induced acinar cell damage leading to death was observed, followed by a faster tissue recovery, Thus the proliferative stimulus on acinar cells may accelerate the unmasking of latent lethal damage, resulting in the earlier replacement of dead cells by new, functionally intact cells. (C) 1995 by Radiation Research Societ