Apoptosis as a predictor of paclitaxel-induced radiosensitization in human tumor cell lines.

Paclitaxel is a deterpene with antitumor activity against a variety of human neoplasms. Paclitaxel cytotoxicity is thought to derive mainly from a stabilization of microtubules as a result of enhanced tubulin polymerization that leads to an accumulation of cells in the mitotic (M) phase of the cell cycle. Because cells in this phase of the cell cycle are known to be radiosensitive, it was thought that paclitaxel, in addition to its direct toxicity, may also sensitize tumor cell populations to radiation. Studies evaluating the radiosensitizing potential of paclitaxel in cultured cells have been equivocal, with only approximately 50% of the tested cell lines showing radiosensitization. To explain this variability, we advanced the hypothesis that the ability of paclitaxel to radiosensitize cells may be inversely correlated to the efficiency with which it induces apoptosis. To test this hypothesis, we studied paclitaxel-induced apoptosis and radiosensitization in seven human tumor cell lines. Approximately one-half of these cell lines showed radiosensitization that was associated with a low apoptotic index (<20% after a 48-h treatment with 10 or 20 nM paclitaxel). The results suggest that the level of apoptosis, after paclitaxel treatment, may predict for paclitaxel-induced radiosensitization, and that it could be introduced as a parameter for the optimization of combined treatment protocols.

Protection from procarbazine-induced testicular damage by hormonal pretreatment does not involve arrest of spermatogonial proliferation.

Hormone treatments that suppress sperm production enhance the recovery of spermatogenesis after gonadal exposure to various cytotoxic agents. It has generally been assumed that the mechanism of protection involved an arrest of spermatogonial kinetics. To test this hypothesis critically, we examined spermatogonial kinetics and numbers in rats in which the completion of spermatogenesis was suppressed with a 6-week testosterone plus 17beta-estradiol treatment that protected the testis from procarbazine-induced damage. Histological examination showed that the numbers of A-aligned, intermediate, and B spermatogonia and preleptotene spermatocytes and their mitoses were unaffected by testosterone plus 17beta-estradiol treatment. Flow cytometric analysis of bromodeoxyuridine-labeled cells showed that the percentage of diploid cells undergoing DNA synthesis, the progression of B spermatogonia and preleptotene spermatocytes through S-phase, the division of intermediate and B spermatogonia, the entry of intermediate spermatogonia into their next S-phase as type B cells, and the progression of cells through meiotic prophase were either unchanged or very slightly increased. Thus, changes in spermatogonial numbers or suppression of their proliferation cannot account for protection of spermatogenesis from exposure to cytotoxic agents.

Hormonal protection from cyclophosphamide-induced inactivation of rat stem spermatogonia.

Studies of protection of testicular function from cyclophosphamide with hormonal pretreatment have been limited by the lack of a convenient model for cyclophosphamide-induced inactivation of stem spermatogonia. In the rat, the mortality from cyclophosphamide had prevented the administration of sufficient dosages to produce detectible damage to stem spermatogonia. To overcome this problem, we used bone marrow transplantation and sodium 2-mercaptoethanesulfonate (Mesna) treatment to raise the lethal dose for 50% of the animals (LD50) for cyclophosphamide from 275 to > 400 mg/kg body weight. In addition we used irradiation, 2 weeks prior to injection of cyclophosphamide, to greatly enhance the measured toxicity of cyclophosphamide towards stem spermatogonia. Whereas sperm counts at 9 weeks after a 300 mg/kg cyclophosphamide dose were reduced by only a factor of 1.6 without prior irradiation, they were reduced by a factor of 60 when 2.5 Gy of irradiation had been given. Dramatic protection against this toxicity was produced by hormone treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Nal-Glu) and an antiandrogen (flutamide) following the radiation but prior to cyclophosphamide. This hormone treatment did not modify the stem cell toxicity of the radiation and it therefore must be protecting stem cells against cyclophosphamide-induced damage. Because GnRH antagonist-antiandrogen treatment can protect stem spermatogonial survival and/or function in the rat from cyclophosphamide-induced damage, if the same principles are applicable in human, hormonal pretreatment should be useful for preventing the prolonged azoospermia caused by chemotherapy with cyclophosphamide-containing protocols.

Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol.

The kinetics of taxol-induced mitotic arrest and apoptosis in murine mammary carcinoma MCA-4 and ovarian carcinoma OCA-I tumors were determined to establish a possible causative relationship between mitotic arrest and apoptosis and to see whether these cellular effects of taxol would correlate with the extent of its antitumor efficacy. Mice bearing 8-mm tumors in a hind leg were given taxol i.v. at a dose of 10-80 mg/kg. Both tumors responded to taxol by significant growth delay or transient regression; in general, the response was greater as the dose of taxol was increased. For kinetics studies the mice were treated with 60 mg/kg taxol given once when tumors were 8 mm in size or twice, with the second dose being given 3 days after the first. At various times ranging from 1 to 96 h after treatment with taxol, tumors were histologically analyzed to quantify mitotic and apoptotic activity. After a single dose of taxol, mitotic arrest was visible at 1 h, and the mitotic index increased with time to reach peak values of 36% in MCA-4 tumors and 22% in OCA-I tumors at 9 h. The index then declined to a baseline of 1%-3% at 3 days for MCA-4 tumors and 1 day for OCA-I tumors. Apoptosis followed mitotic arrest, beginning at the time of peak mitotic arrest, increasing to the highest level of about 20% at 18-24 h after treatment and gradually declining to the normal level of 3%-6% after 3-4 days. Nuclear material progressively condensed in mitotically arrested cells, culminating in the frank appearance of multiple apoptotic bodies. The change in cell morphology plus the dynamics of apoptosis development imply that a large percentage of tumor cells arrested in mitosis by taxol die by apoptosis. Kinetic analysis undertaken after the second dose of taxol showed a considerably lower percentage of cells arrested in mitosis as compared with that seen after a single dose, and the induction of apoptosis by the second dose was minimal. However, the antitumor efficacy of the second dose of taxol was similar to or better than that of the first dose, implying that in addition to mitotic arrest and apoptosis, there exist other mechanisms by which taxol exerts its antitumor action.

Protection from radiation-induced damage to spermatogenesis by hormone treatment.

Infertility caused by killing of the spermatogonial stem cells occurs frequently in men treated for cancer with radiotherapy and chemotherapy. We investigated whether pretreatment of rats with testosterone plus estradiol, which reversibly inhibits the completion of spermatogenesis and protects spermatogonial stem cells from procarbazine-induced damage, would also protect these cells from radiation. Adult male LBNF1 rats were implanted for 6 weeks with capsules containing testosterone and estradiol and then irradiated with doses from 2.5-7.0 Gy. Controls were irradiated with 1.8-3.5 Gy. Implants were removed 1 day after irradiation, and all animals were killed 10 weeks later for assessment of stem cell survival by counting repopulating tubules in histological sections and by sperm head counts. At doses of 2.5 and 3.5 Gy the repopulation indices and sperm head counts were significantly higher (P < 0.001) in the rats treated with testosterone and estradiol than in the controls. Protection factors (dose-modifying factors) calculated from the dose-response curves were in the range of 1.5-2.2. Elucidation of the mechanism of protection is essential to apply it to clinical situations. The fact that the spermatogonia are protected against radiation as well as procarbazine indicates that the mechanism does not involve drug delivery or metabolism.

Enhancement of tumor radioresponse of a murine mammary carcinoma by paclitaxel.

Paclitaxel is a chemotherapeutic agent with potent microtubule stabilizing activity that arrests cells in G2-M. Because G2 and M are the most radiosensitive phases of the cell cycle, paclitaxel has potential as a cell cycle-specific radiosensitizer. In this study, we investigated the ability of paclitaxel to increase tumor radioresponse in vivo using a murine mammary carcinoma and the dependency of this response on accumulation of tumor cells in mitosis. Mice bearing 8-mm tumors were treated with paclitaxel (60 mg/kg i.v.), 9, 15, or 21 Gy of single-dose radiation, or with a regimen of both agents in which radiation was given 1, 9, or 24 h after paclitaxel. The effect of the treatments was determined by tumor growth delay. Microscopically, the percentage of mitotically arrested cells was only 4% 1 h after treatment with paclitaxel, increased to a maximum value of 30% at 9 h, and decreased to 12% 24 h after paclitaxel. Paclitaxel enhanced tumor radioresponse by factors of 1.21 to 2.49. The degree of enhancement increased with increases in both the dose of radiation and the time between paclitaxel administration and radiation delivery. Radiation efficiently destroyed mitotically arrested cells by apoptosis. The greatest enhancement of radiation response was not at the time of the highest mitotic arrest but at 1 day after paclitaxel treatment, showing that paclitaxel potentiates tumor radioresponse by mechanisms in addition to blocking the cell cycle in mitosis, possibly by tumor reoxygenation. Thus, these results show that paclitaxel is a potent in vivo radiopotentiating agent and has the potential to be usefully combined with radiotherapy.

Hormonal protection from procarbazine-induced testicular damage is selective for survival and recovery of stem spermatogonia.

Procarbazine produces long-term sterility in the male by killing stem spermatogonia. The degree and selectivity of protection of stem spermatogonia in rats from procarbazine by pretreatment with steroid hormones were investigated. Male LBNF1 rats were treated for 6 weeks with Silastic implants containing testosterone plus 17 beta-estradiol. The hormone-treated rats and sham-treated controls were given a single injection of graded doses of procarbazine and the hormone implants were removed the next day. Spermatogonial stem cell survival and function, assessed by the repopulation indices and sperm head counts 10 weeks later, showed that stem spermatogonia were protected by testosterone plus 17 beta-estradiol treatment from the toxic effects of procarbazine with a dose-modifying protection factor of about 2.5. In contrast, there was no hormonal protection from the procarbazine-induced killing of differentiating spermatogonia, preleptotene spermatocytes, and spermatocytes in meiotic prophase or from the delay in maturation of round spermatids, assessed 9 days after procarbazine injection by histological or flow cytometric methods. In addition, there was no hormonal protection from the procarbazine-induced decline in body weights and lymphocyte counts, indicating that the gastrointestinal, neurological, and hematological systems were not protected. The specificity of protection indicates that the hormonal protection of the stem spermatogonia is not the result of a systemic or overall testicular decrease in drug delivery, decrease in bioactivation, nor increase in drug detoxification, except possibly within the stem cells themselves. We conclude that the degree of hormonal protection and its specificity would be appropriate for clinical application provided that the mechanism of protection is elucidated and appears applicable to humans.