A phase 3b study of sofosbuvir plus ribavirin in treatment-naive and treatment-experienced Korean patients chronically infected with genotype 2 hepatitis C virus.

In Korea, patients with chronic hepatitis C virus (HCV) infection are typically treated with pegylated interferon-alpha plus ribavirin, but interferons are contraindicated in many patients and are often poorly tolerated, particularly by the elderly and those with advanced liver disease. No interferon-free treatment regimens are approved in Korea. Sofosbuvir is an oral nucleotide analog inhibitor of the HCV nonstructural 5B RNA polymerase. It is approved in the USA, European Union and Japan for treating a number of HCV genotypes, including genotype 2. Genotype 2 has a seroprevalence of 38-46% in Korea. This single-arm, phase 3b study (NCT02021643) examined the efficacy and safety of sofosbuvir plus ribavirin (12-week duration) in chronic genotype 2 HCV-infected treatment-naive and treatment-experienced Korean patients with and without cirrhosis. The proportion of patients with sustained virologic response 12 weeks after treatment discontinuation (SVR12) was 97% (125/129), with 96% (101/105) of treatment-naive and 100% (24/24) of treatment-experienced patients achieving SVR12. Two patients experienced virologic failure (n = 1, on-treatment failure; n = 1, relapse). No patient discontinued study treatment due to an adverse event (AE). The most common treatment-emergent AEs were headache (18%, 23/129) and pruritus (15%, 19/129). Few patients had grade 3 AEs (5%, 6/129) or grade 3 laboratory abnormalities (12%, 15/129). No grade 4 AE was reported. These data suggest that 12 weeks of treatment with the all-oral, interferon-free regimen of sofosbuvir plus ribavirin is effective and well tolerated in Korean patients with chronic genotype 2 HCV infection.

Sofosbuvir plus ribavirin for the treatment of patients with chronic genotype 1 or 6 hepatitis C virus infection in Hong Kong.

BACKGROUND: In Hong Kong, most patients with hepatitis C virus (HCV) have either genotype 6a or 1b infection. AIM: To evaluate the efficacy and safety of sofosbuvir with ribavirin in treatment-naïve patients in Hong Kong with HCV genotype 1 or 6. METHODS: In an open-label study, patients were randomised to sofosbuvir 400 mg once daily plus ribavirin 1000-1200 divided twice daily for 12 (n = 10), 16 (n = 11) or 24 (n = 10) weeks. The primary endpoint was the percentage of patients with HCV RNA < LLOQ (lower limit of quantification, 25 IU/mL) 12 weeks after cessation of therapy (SVR12). RESULTS: All 31 patients (20 HCV genotype 1 and 11 genotype 6) had HCV RNA < LLOQ by Week 4 of treatment and at their last on-treatment visit. SVR12 rates were high in all treatment groups: 100% (10/10) for 12 weeks, 100% (11/11) for 16 weeks and 90% (9/10) for 24 weeks of therapy. The only patient who did not reach SVR12 had genotype 1 HCV and relapsed at post-treatment Week 4. Sofosbuvir with ribavirin was generally well tolerated. The most common adverse events were malaise (13%) and upper respiratory tract infection (13%), followed by anaemia (10%). No patients experienced serious adverse events. One patient discontinued treatment at Week 16 because of an adverse event. The event, upper respiratory tract infection, was not considered treatment related by the investigator. This subject achieved SVR12. CONCLUSIONS: The all-oral regimen sofosbuvir plus ribavirin is effective in treatment-naïve patients in Hong Kong with genotype 1 or 6 HCV. TRIAL REGISTRATION NUMBER: NCT02021643.

Tumor-selective killing by selenite in patient-matched pairs of normal and malignant prostate cells.

BACKGROUND: Selenium compounds have been shown to induce apoptosis in a variety of human prostate cancer cell lines. However, the effects of selenium have yet to be examined in normal and malignant cells derived from the same individual. Selenite metabolism consumes glutathione (GSH) and produces superoxide. The generation of reactive oxygen species is an important mechanism in selenite-induced apoptosis. METHODS: Three patient-matched pairs of primary prostatic epithelial cell cultures from normal and cancer were evaluated for their response to selenite. Apoptosis was measured and the differential response of normal and cancer cells was correlated with the expression of bcl-2, bax, GSH, and manganese superoxide dismutase (MnSOD). RESULTS: The cancer-derived cells were significantly more sensitive to selenite-induced apoptosis than the corresponding normal cells. Tumor-selective killing was not observed in cells treated with selenomethionine. The ratio of bcl-2:bax was decreased in the cancer-derived cells treated with selenite. Total GSH concentrations were similar in paired normal and cancer cells. Therefore, differences in GSH content do not appear to play a role in tumor-selective killing by selenite. Superoxide is a by-product of selenite metabolism and normal cells showed increased MnSOD expression and SOD activity compared to the cancer-derived cells. Prostate cancer cells treated with the MnSOD mimetic, MnTMPyP, were protected against the cytotoxic effects of selenite. CONCLUSIONS: Higher MnSOD expression in normal cells may play an important role in eliminating superoxide radicals produced as a result of selenite metabolism and contribute to the tumor-selective killing by selenite in prostate cancer.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line.

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.

Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-Hodgkin's lymphomas.

PURPOSE: To evaluate the efficacy and safety of tositumomab and iodine I 131 tositumomab (Bexxar; Corixa Corp, Seattle, WA, and GlaxoSmithKline, Philadelphia, PA) in patients with chemotherapy-refractory low-grade or transformed low-grade non-Hodgkin's lymphoma (NHL) and to compare its efficacy to the patients' last qualifying chemotherapy (LQC) regimens. PATIENTS AND METHODS: Sixty patients who had been treated with at least two protocol-specified qualifying chemotherapy regimens and had not responded or progressed within 6 months after their LQC were treated with a single course of iodine I 131 tositumomab. RESULTS: Patients had received a median of four prior chemotherapy regimens. A partial or complete response (CR) was observed in 39 patients (65%) after iodine I 131 tositumomab, compared with 17 patients (28%) after their LQC (P <.001). The median duration of response (MDR) was 6.5 months after iodine I 131 tositumomab, compared with 3.4 months after the LQC (P <.001). Two patients (3%) had a CR after their LQC, compared with 12 (20%) after iodine I 131 tositumomab (P <.001). The MDR for CR was 6.1 months after the LQC and had not been reached with follow-up of more than 47 months after iodine I 131 tositumomab. An independent review panel verified that 32 (74%) of the 43 patients with nonequivalent durations of response (> 30 days difference) had a longer duration of response after iodine I 131 tositumomab (P <.001). Only one patient was hospitalized for neutropenic fever. Five patients (8%) developed human antimurine antibodies, and one (2%) developed an elevated TSH level after treatment. Myelodysplasia was diagnosed in four patients in follow-up. CONCLUSION: A single course of iodine I 131 tositumomab was significantly more efficacious than the LQC received by extensively pretreated patients with chemotherapy-refractory, low-grade, or transformed low-grade NHL and had an acceptable safety profile.

Long-term follow-up of patients with Stage III follicular lymphoma treated with primary radiotherapy at Stanford University.

PURPOSE: To report the long-term survival and late toxicity data of Stage III follicular lymphoma patients treated with primary radiotherapy. METHODS AND MATERIALS: Sixty-six patients with Stage III follicular small cleaved (FSC) or follicular mixed (FM) non-Hodgkin's lymphoma were treated with total lymphoid irradiation (61 patients) or whole body irradiation (5 patients) as their primary treatment modality from 1963 to 1982 at Stanford University. Adjuvant chemotherapy was given to 13 patients. RESULTS: Median follow-up was 9.5 years with a range of 0.5-24.3 years. Median overall survival, cause-specific survival, freedom from relapse, and event-free survival were 9.5, 18.9, 7.1, and 5.1 years, respectively. Few initial relapses or lymphoma-related deaths were seen beyond the first decade of follow-up. Patient age and number of disease sites were the two strongest predictors of overall survival. The cohort of patients with limited Stage III disease demonstrated an 88% freedom from relapse and a 100% cause-specific survival with up to 23.5 years follow-up. CONCLUSION: The long-term survival data for Stage III FSC or FM non-Hodgkin's lymphoma treated with primary radiotherapy are at least comparable and possibly better than results achieved with other therapeutic approaches. Patients with limited Stage III disease do particularly well. Whether these results are superior to an initial approach of deferred therapy until clinically indicated is currently unknown.

Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization.

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.

Radioimmunotherapy of B-cell NHL.

Much progress has been made in the development and implementation of radionuclide-carrying antibody therapy (radioimmunotherapy or RIT) of non-Hodgkin's lymphomas (NHL) in the past decade. Response rates have generally exceeded 60% for nonmyeloablative single dose RIT (85% - 100% for myeloablative) in patients who have relapsed after primary therapy. It is also encouraging that the duration of such responses has often been greater than the response to the last chemotherapeutic regimen administered. These results, as well as a favorable toxicity profile, have resulted in the successful earlier and more widespread use of this new therapeutic modality. Although unlabeled antibody therapy alone has had a positive impact on the treatment of NHL, the response rates for RIT have been higher than (sometimes nearly double) those for unlabeled antibody therapy. This has been demonstrated in trials that have directly compared radiolabeled antibody with unlabeled antibody, as well as in separate trials for similar patient groups. Use of radionuclides in conjunction with antibodies adds transient marrow suppression and a small risk of second malignancy over unlabeled antibody therapy. However, the toxicity from a single course of RIT is very favorable compared to chemotherapy. Despite the enormous progress of RIT, much remains to be learned to fully optimize the role of this exciting modality in the treatment of NHL.

Radiation induces upregulation of cyclooxygenase-2 (COX-2) protein in PC-3 cells.

PURPOSE: To investigate the impact of gamma-irradiation on cyclooxygenase-2 (COX-2) expression and its enzymatic activity in PC-3 cells. Cell cycle redistribution, viability, and apoptosis were quantitated in control and irradiated cells with or without the COX-2 inhibitor NS-398. METHODS AND MATERIALS: Western blot analysis was used to assess COX-2 protein expression. Prostaglandin (PGE(2)) was measured after addition of arachidonic acid (AA) using a Monoclonal Immunoassay Kit. Cell cycle and apoptosis were assessed using flow cytometry. RESULTS: We observed a dose-dependent increase in COX-2 of 37.0%, 79.7%, and 97.5% following irradiation with 5, 10, and 15 Gy, respectively. The PGE(2) level of irradiated cells was higher than in controls (1512 +/- 157.5 vs. 973.7 +/- 54.2 rhog PGE(2)/mL; p < 0.005, n = 4) while cells irradiated in the presence of NS-398 had reduced PGE(2) levels (218.8 +/- 80.1 rhog PGE(2)/mL; p < 0.005; n = 4). We found no differences in cell cycle distribution or apoptosis between cells irradiated in the presence or absence of NS-398. CONCLUSIONS: COX-2 protein is upregulated and enzymatically active after irradiation, resulting in elevated levels of PGE(2). This effect can be suppressed by NS-398, which has clinical implications for therapies combining COX-2 inhibitors with radiation therapy.

Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis.

Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.

Clinical radioimmunotherapy.

Radioimmunotherapy (RIT) is a promising new therapy for the treatment of a variety of malignancies. General principles of RIT are discussed, including important considerations in the selection of monoclonal antibodies (MAb) and radionuclides for RIT. Results of clinical trials using RIT for the treatment of lymphoma, leukemia, and solid tumors are summarized. The results from many of these trials are promising, especially for the treatment of lymphohematopoietic malignancies, in which a variety of MAb, radionuclides, and study designs have resulted in high response rates with a number of durable responses. Encouraging results have also been obtained using RIT to treat some solid tumors, primarily in patients with relatively low tumor burdens. RIT is generally well tolerated, with the primary toxicity being transient reversible myelosuppression in most nonmyeloablative studies. Nonhematologic toxicity, especially at nonmyeloablative doses, has been minimal in most studies. Approaches for increasing the therapeutic index of RIT are reviewed, which may further potentiate the efficacy and decrease the toxicity of RIT.

Multicenter phase II study of iodine-131 tositumomab for chemotherapy-relapsed/refractory low-grade and transformed low-grade B-cell non-Hodgkin's lymphomas.

PURPOSE: This multicenter phase II study evaluated the efficacy, dosimetry methodology, and safety of iodine-131 tositumomab in patients with chemotherapy-relapsed/refractory low-grade or transformed low-grade non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Patients received a dosimetric dose that consisted of 450 mg of anti-B1 antibody followed by 35 mg (5 mCi) of iodine-131 tositumomab. Serial total-body gamma counts were then obtained to calculate the patient-specific millicurie activity required to deliver the therapeutic dose. A therapeutic dose of 75 cGy total-body dose (attenuated to 65 cGy in patients with platelet counts of 101,000 to 149,000 cells/mm(3)) was given 7 to 14 days after the dosimetric dose. RESULTS: Forty-five of 47 patients were treated with a single dosimetric and therapeutic dose. Twenty-seven patients (57%) had a response. The response rate was similar in patients with low-grade (57%) or transformed low-grade (60%) NHL. The median duration of response was 9.9 months. Fifteen patients (32%) achieved a complete response (CR; 10 CRs and five clinical CRs), including five patients (50%) with transformed low-grade NHL. The median duration of CR was 19.9 months, and six patients have an ongoing CR. Treatment was well tolerated, with the principal toxicity being hematologic. The most common nonhematologic toxicities that were considered to be possibly related to the treatment included mild to moderate fatigue (32%), nausea (30%), fever (26%), vomiting (15%), infection (13%), pruritus (13%), and rash (13%). Additionally, one patient developed human-antimouse antibodies. CONCLUSION: Iodine-131 tositumomab produced a high overall response rate, and approximately one third of patients had a CR despite having chemotherapy-relapsed or refractory low-grade or transformed low-grade NHL.

Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer.

A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (90Y-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m2 (mean administered dose, 106.5 +/- 10.3 mCi/m2) of 90Y-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered approximately 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m2 90Y. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs.

Radiosensitization by intratumoral administration of cisplatin in a sustained-release drug delivery system.

PURPOSE: Effects of combining local irradiation and intratumoral (i.t.) administration of cisplatin (CDDP) in a sustained-release drug delivery system (epi gel) were studied in a murine SCCVII squamous cell carcinoma model in mice. MATERIALS AND METHODS: The epinephrine injectable gel was used as a drug delivery system. Intratumoral pharmacokinetics of CDDP was studied by using 195mPt-CDDP. The tumor volume quadrupling time (TVQT) and tumor growth delay (TGD) time were used to evaluate the antitumor efficacy of treatment regimens. RESULTS: The concentration and residence of 195mPt-CDDP was significantly higher in tumors treated with 195mpt-CDDP/epi gel than in tumors treated with 195mPt CDDP gel or 195mPt-CDDP suspension. Intratumoral administration of CDDP/epi gel (4 mg/kg) produced an average TGD time of 15.5 +/- 2.8 days, which was 5.2 - 7.4 times longer than CDDP suspension i.t. or i.p. When combined with a single dose of radiation (10 Gy), i.t. administration of CDDP/epi gel was 2.0 - 3.6-fold as effective as administered i.t. in suspension (39.2 +/- 4.1 vs. 19.8 +/- 3.9 days of TGD, P < 0.05) or i.p. in solution (39.2 +/- 4.1 vs. 11.0 +/- 1.6 days, P < 0.001) in inhibiting tumor growth and produced 20-60% complete remission of tumors. When combined with fractionated irradiation, pre-irradiation CDDP administration was more effective than post-radiation administration (26.7 vs. 12.1 days of TGD, P < 0.05). Mice treated with CDDP/epi gel i.t. alone or in combination with irradiation, had little systemic toxicity. CONCLUSIONS: Intratumoral administration of CDDP using the sustained-release drug delivery system is an efficient and safe method to maximize the drug concentration in tumor, minimize the systemic toxicity and enhance antitumor efficacy of irradiation.

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.

The role of radiation-induced apoptosis as a determinant of tumor responses to radiation therapy.

Ionizing radiation is an effective means of killing tumor cells. Approximately 50% of all American cancer patients are treated with radiotherapy at some time during the course of their disease, making radiation one of the most widely used cytotoxic therapies. Currently, much effort is focused on understanding the molecular pathways that regulate tumor cell survival following radiotherapy, with the long term goal of developing novel therapeutic strategies for specifically sensitizing tumors to radiation. At present, there is particular interest in the role of tumor cell apoptotic potential as a regulator of both intrinsic and extrinsic determinants of the response of tumors to radiation therapy. Here we review what is currently known about the role of apoptosis as a mechanism of tumor cell killing by ionizing radiation and the relative contribution of apoptosis to cellular radiosensitivity and the ability to control human cancers using radiotherapy. The following topics will be discussed: (1) radiation-induced apoptosis in normal and malignant cells, (2) clinical findings with respect to apoptosis in human cancers treated with radiotherapy, (3) the contribution of apoptosis to intrinsic radiosensitivity in vitro, (4) the relevance of apoptosis to treatment outcome in experimental tumor models in vivo and (5) the potential of exploiting apoptosis as a means to improve the therapeutic efficacy of radiotherapy.

Myc activation reduces fibroblast clonogenicity via an apoptotic mechanism that can be suppressed by a soluble paracrine factor.

The c-Myc transcription factor is involved in the regulation of cellular proliferation and differentiation and is one of the most frequently deregulated genes in human cancers. While c-Myc is known to enhance the proliferative potential of cells, its activation in immortalized fibroblasts has been found to result in apoptosis following gamma-irradiation or under adverse growth conditions, including serum deprivation and hypoxia. When plating Rat-1 fibroblasts at low cell densities (100 cells/100 mm plate), we observed a substantial reduction in the clonogenicity of cells with deregulated c-Myc activity compared to cells with normal c-Myc activity. This difference in clonogenicity was apparent despite the fact that cells were plated in media containing sufficient serum and oxygen concentrations known to suppress apoptosis of exponentially growing Rat-1 fibroblasts with activated c-Myc. Therefore, we hypothesized that the observed reduction in plating efficiency in cells with activated c-Myc occurred via an apoptotic mechanism and that a fibroblast-derived factor was required for suppression of apoptosis. Overexpression of the anti-apoptotic oncogene, Bcl-2, in cells with activated c-Myc restored the plating efficiency to normal levels in cells plated at low cell densities. This strongly suggested that the decreased clonogenicity of fibroblasts with altered c-Myc activity resulted from enhanced apoptosis of the cells under these conditions. Furthermore, plating cells on a feeder layer of lethally-irradiated fibroblasts or in Rat-1 conditioned media increased the plating efficiencies of sparsely plated cells in a dose-dependent fashion. These results suggest that in addition to previously reported requirements for serum-derived growth factors and normal oxygen conditions, a paracrine factor liberated by Rat-1 fibroblasts is required to suppress c-Myc-induced apoptosis in these cells.

Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy.

Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.

Effects of keratinocyte growth factor on the proliferation and radiation survival of human squamous cell carcinoma cell lines in vitro and in vivo.

PURPOSE: Keratinocyte growth factor (KGF) has potent mitogenic activity on normal epithelial cells and has been found to enhance intestinal crypt cell survival in irradiated mice and to prevent radiation and chemotherapy-induced mucositis in animal models. The purpose of the study reported here is to investigate the effect of recombinant human KGF on the proliferation and survival of human squamous carcinoma cell lines following irradiation. METHODS AND MATERIALS: The level of KGF receptor (KGFR) mRNA in normal Balb/Mk cell line and human head and neck squamous carcinoma cell lines was assessed using a RNase protection assay. The clonogenic assay and MTT assay were used to study the proliferative effects of KGF on human tumor cell lines and Balb/MK cell line in vitro. Effects of KGF on in vivo tumor growth and radiosensitivity were studied in three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice. RESULTS: Seven of 10 tumor cell lines studied expressed KGFR mRNA. None of these tumor cell lines showed enhanced proliferation when exposed to KGF for 2 days or less. Prolonged exposure to KGF for 7 days or longer resulted in low level stimulation of proliferation in both clonogenic and MTT assays in four of seven KGFR-positive cell lines. Two KGFR-negative cell lines also had a low proliferative response to KGF in a clonogenic assay, but not in the MTT assay. Normal keratinocyte Balb/MK cells, which expressed a moderate level of KGFR mRNA, had a strongly proliferative response to KGF. Its KGF enhancement ratio (KER) of plating efficiency was 24-70 times higher than that of the tumor cells studied (p < 0.001). The KGF-stimulated tumor cell growth was almost completely inhibited by heparin or epidermal growth factor (EGF). There were no significant differences (p > 0.05) in the survival of any of tumor cell lines in the presence or absence of KGF (100 ng/ml) irradiated with doses of 0-15 Gy, and no significant differences (p > 0.05) between the radiobiological parameters D0, Dq, and n number from the SHMT model, alpha, beta, and alpha/beta ratio from the LQ model and SF2 for radiation survival curves for cell lines irradiated in the presence or absence of KGF. Three KGFR-positive human squamous cell carcinoma xenografts in nude mice, and a murine KGFR-negative melanoma tumor in Balb/c mice treated with 1.0 mg/kg of KGF for 3 days grew at the same rate as in untreated mice. CONCLUSION: The recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of cancers.