Growth of mammalian multicellular tumor spheroids.

The in vitro growth of small (0.05 to 3 mm diameter) avascular multicellular tumor spheroids from six rodent and two human tumor lines has been analyzed. Surprisingly, the radial increase of multicellular tumor spheroids is linear with time after a brief initial period of geometric growth. These multicellular tumor spheroids are shown to have a constant thickness of proliferative outer crust and of middle nonproliferative but viable mantle. An analytical model for their growth is developed which explains the growth pattern. This constant crust thickness model leads to a progressively diminishing growth fraction as radius increases and should be applicable to such early growth of micrometastases in vivo. The model also provides a procedure for determining cell cycle time.

Transplacental uptake of WR 2721 by the rat embryo.

On day 14 post-conception, near the end of the period of major organogenesis, pregnant rats were injected intravenously or intraperitoneally with WR 2721 spiked with 14C-WR 2721. The radioprotectant was shown to cross the placenta rapidly when administered by either route, and the concentration of WR 2721 in the embryos, placentae, and maternal blood plasma was determined during the period 5 to 90 minutes following administration. The concentration of WR 2721 increased continuously in the embryos during this period and did so against a decreasing concentration in the maternal blood. Injection of WR 2721 at 100 mg/kg of maternal body weight resulted in the presence of 8-9-mg/kg embryo weight; this embryo level is about 1/2 the injected dose of WR 2721 currently being used in human radiotherapy trials, that is, 20 mg/kg (740 mg/m2) body weight. Previous toxicity studies of 9, 11, and 14 day rat embryos have shown that this 100 mg/kg dose is much below the level which produces embryotoxic effects.

Isoproterenol in comparison to WR-2721 as a chemoradioprotector of the rat parotid gland.

A comparison was made of the radioprotective abilities of the chemoradioprotector WR-2721, an amino-thiol, and the beta-sympathomimetic secretogogue isoproterenol on the rat parotid gland. Using the dose-response curve of WR-2721 for gland weights as a basis for comparison, isoproterenol was found to offer significant and equal protection during both the acute (DMF, 2.5) and the chronic (DMF, 2.3) periods.

Radioprotection of the rat parotid gland by cAMP.

Most earlier studies showing a radioprotective effect by cAMP show only slight degrees of protection. The present study demonstrates a substantial protective effect (DMF, 1.63) of exogenously administered cAMP on the rat parotid gland and supports the mechanism suggested previously for protection afforded the parotid glands by the beta-adrenergic agonist isoproterenol, which is known to elevate endogenous intracellular cAMP.

Radioprotection of the rat parotid gland by WR-2721 and isoproterenol and its modification by propranolol.

The aminothiol WR-2721 and beta-adrenergic agonist isoproterenol both conferred considerable radioprotection to the rat parotid gland. Isoproterenol acts on the beta-receptor, and its specific antagonist, propranolol, eliminated isoproterenol's protective effect, implicating the beta-receptor and possibly cAMP in the mechanism of the protection. Since other sulfhydryl-containing protectants have been shown to elevate cAMP it was reasoned that WR-2721 might do so as well. However, the radioprotection conferred by WR-2721 was not reduced by propranolol, showing that the beta-receptor played no part in WR-2721's action. The possible role of cAMP in radioprotection by isoproterenol is discussed.

Comparison of cAMP with other radioprotectors against chronic damage to the rat parotid gland.

Radiation damage to the parotid gland is protectable by cAMP during the first week after irradiation (acute phase), though appreciable recovery occurred later with or without such protection. Further damage developed later (chronic phase, 60-90 days), and cAMP was still protective against this damage with a dose modification factor of 1.86 for gland weight. A summary of the protective factors, acute and chronic, for WR-2721, isoproterenol, and cAMP is included. Chronic damage is about 1.5 times as great as acute, and protection against acute and chronic damage is about equal for all three compounds.

Effect of WR-2721 on fetal development in the rat.

The radioprotector WR-2721 has been shown to radioprotect all tissues studied except the central nervous system. However, it has not yet been used to radioprotect the fetus. In this study we determined that [14C]WR-2721 injected into pregnant rats quickly passed the placenta and was concentrated by the fetus. In addition, we evaluated the toxicity of WR-2721 (2.5-600 mg/kg) to pregnant rats and their fetuses during the period of major organogenesis at Days 9, 11, and 14 postconception. Pregnant animals were only slightly more (10%) sensitive (LD50, 580 mg/kg) to WR-2721 than nonpregnant cohort animals (LD50, 640 mg/kg). At concentrations of 50 mg/kg or less there was a small but statistically significant increase in fetal deaths, while at doses greater than 300 mg/kg a larger degree of fetal mortality occurred. Maximal fetal weight loss, to about 84% of control, was found at 500 mg/kg. No changes in head dimensions or gross malformations of the surviving fetuses were detected at any time or concentration. Of all the parameters measured in this study none demonstrated a predilection for any specific period of major organogenesis. The results of this study indicate that while WR-2721 demonstrates a dose-related embryotoxicity it is not teratogenic.

WR-2721 entry into the brain across a modified blood-brain barrier.

Radioprotection of the CNS by WR-2721 has not been possible because of its inability to cross the blood-brain barrier (BBB) and so gain access to the neural tissue. Modification of the BBB using hypertonic arabinose (1.8 m), injected via the internal carotid artery (ica), permitted entry of ip-injected [14C]WR-2721 into the ipsilateral cerebral hemisphere. The BBB-modified hemisphere had a 5.34-fold increased uptake compared to nonmodified controls. Delivery as a bolus via the ica further enhanced uptake after BBB opening; WR-2721 was 3.73 times greater than by ip injection. A 20-fold increase of WR-2721 brain uptake has been calculated for ica administration with the BBB opened as compared to the ip route without BBB modification. Toxicity of ip-administered WR-2721 with the BBB open was only 1.4 times greater than non-modified controls and 1.96 times more toxic when delivered via the ica. These data demonstrate significant uptake of WR-2721 into the CNS, a previously unprotected organ, and provide a model for future radioprotective studies.

Early changes accompanying development of adriamycin resistance in spheroids of a mouse mammary carcinoma line.

The early changes accompanying development of ADR resistance were studied in a mouse mammary adenocarcinoma cell line sensitive to ADR (S). Resistant cells (R) were derived from S by five separate exposures to 1 microgram/ml ADR for 1 hr. The R cells had a 3-fold increased resistance to ADR, a higher proportion of larger cells, higher numbers of chromosomes per cell and higher incidence of minute chromosomes. R spheroids had a higher degree of differentiation. They were more compact, had increased numbers of gap-junctions, and altered membranes. An inversion in the X chromosome in S cells was corrected in the R cells.