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.

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.

p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts.

Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.

Modulation of c-Myc activity and apoptosis in vivo.

We have developed an animal tumor model system to study the effects of c-Myc activation on apoptosis induction in vivo. Tumors were generated in SCID mice from Rat-1 fibroblasts that constitutively express an inactive c-Myc-estrogen receptor fusion protein (T.D. Littlewood et al, Nucleic Acids Res., 23: 1686 -1690, 1995), which is activated in vivo by the administration of 4-hydroxytamoxifen in time release pellets. We demonstrate that activation of c-Myc results in a substantial increase in the number of apoptotic tumor cells and that this apoptosis is predominant in regions of tumor hypoxia. c-Myc-induced apoptosis of hypoxic cells is inhibited in tumors that overexpress the human Bcl-2 protein. Bcl-2, however, does not prevent p53 protein accumulation or the down-regulation of the cyclin-cdk inhibitor p27 protein following c-Myc activation by 4-hydroxytamoxifen. This result suggests that Bcl-2 does not affect c-Myc function directly but acts downstream of c-Myc to inhibit apoptosis. We propose that the ability of activated c-Myc to enhance cellular proliferation might contribute to the genesis of early neoplasms that are held in check by the alternate ability of c-Myc to induce apoptosis of cells that have outgrown their supply of oxygen or other factors associated with hypoxic regions of solid tumors. Secondary genetic lesions downstream of c-Myc that suppress the apoptotic potential of tumor cells, such as Bcl-2 overexpression, might play an important role in the malignant progression of these tumors because they would disrupt the balance between apoptosis and proliferation initiated by c-Myc deregulation.

Association of BCL-2 with membrane hyperpolarization and radioresistance.

The resting membrane potential of parental, neomycin control, and Bcl-2 transfected cells was measured, and the effect of membrane hyperpolarization or depolarization on radiosensitivity was studied. Bcl-2 transfected cells were significantly more radioresistant than control cells and were significantly hyperpolarized compared to parental and neomycin control transfected PW and HL60 cells. Hyperpolarization of the parental and neomycin control transfected cells by valinomycin significantly increased the radioresistance of these cells to such an extent that there was no longer a significant difference in the survival of the valinomycin treated and irradiated control cells compared to similarly irradiated Bcl-2 transfected cells. In contrast, depolarization of the Bcl-2 transfected PW and HL60 cells decreased the radioresistance of the Bcl-2 transfectants to a level similar to that of the control cells. The data presented here suggest that overexpression of Bcl-2 affects membrane potential and that this hyperpolarization is associated with increased radioresistance of cells that overexpress Bcl-2. Furthermore, Bcl-2 transfected cells were also less susceptible to the specific Na+/K(+)-ATPase inhibitor ouabain, suggesting that Bcl-2 may act at the level of the Na+/K(+)-ATPase pump.

Over-expression of Bcl-2 protects against apoptosis induced by the bioreductive cytotoxic drug SR4233 (Tirapazamine).

The human B-cell lymphoma cell line PW undergoes radiation-induced programmed cell death (PCD). Bcl-2 transfected PW cells, that overexpressed Bcl-2, were significantly more radioresistant than parental or neomycin control transfected PW cells. The viability of Bcl-2 transfected cells was significantly greater than that of parental PW cells treated with the bioreductive cytotoxin SR4233 under aerobic conditions. Bcl-2 transfectants were also significantly more resistant to hypoxia-induced PCD. However, there was no significant difference in the viability of parental and Bcl-2 transfected cells exposed to SR4233 under hypoxic conditions (pO(2)<100 ppm). Incubation of parental PW cells with N-acetyl cysteine decreased the cytotoxicity of SR4233 under aerobic but not anaerobic conditions. Depletion of cellular glutathione with buthionine sulphoxamine killed nearly 100% of control PW cells, but none of the Bcl-2 transfectants under the same conditions. The TBARS assay for lipid peroxidation showed that Bcl-2 transfectants had a significantly lower level of lipid peroxidation than parental PW cells following a 24 hour constant exposure to SR4233 under aerobic conditions. These results suggest that Bcl-2 overexpression inhibits PCD induced by the bioreductive cytotoxin SR4233 under aerobic conditions as well as PCD induced by hypoxia, and that there are other pathways leading to PCD that are unaffected by Bcl-2 overexpression.

Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II beta-subunits using the polymerase chain reaction.

mRNA was isolated from Fundulus heteroclitus pituitaries and used to construct a cDNA library in lambda gt22A. A series of synthetic oligonucleotides, based on conserved regions of teleost gonadotropic hormone (GTH) beta-subunits, were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. Appropriate length PCR products were subcloned and sequenced. Eight clones were eventually identified as cDNAs encoding two distinct beta-subunits of F. heteroclitus, GTH I and GTH II. By comparison with known GTH sequences, putative signal sequences of 19 end 21 amino acids and mature beta-subunits of 95 and 115 amino acids were found for GTH I and GTH II, respectively. Both beta-subunits had well conserved cysteine positions when aligned with other members of the glycoprotein family. The elucidation of the complete nucleotide sequences of two types of F. heteroclitus GTH provides definitive proof that in this species there are at least two distinct forms of pituitary GTH analogous to the classical luteinizing hormone-follicle stimulating hormone family.