Enhancement of photodynamic cell killing (with chloroaluminum phthalocyanine) by treatment of V79 cells with the ionophore nigericin.

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells by photodynamic therapy (PDT), when cells pretreated with 1 microM chloroaluminum phthalocyanine are incubated with nigericin before exposure to red light. Nigericin affects primarily the shoulder of the PDT dose-response curve, reducing the surviving fraction from 0.90 to 0.02 after a fluence of 7 kJ/m2 and from 0.80 to 0.0003 after a fluence of 12 kJ/m2. Optimal enhancement of PDT occurs when cells are incubated with 2 microM nigericin, at pHe 6.7, for 30 to 60 min before irradiation. However, significant enhancement of PDT also occurs when nigericin is added immediately before irradiation. Treatments with chloroaluminum phthalocyanine and nigericin, nigericin alone, or nigericin and red light are not toxic to cells. Cells treated with the combined agents display a rounded morphology 2 h after light exposure and lyse within 12 h. However, rounding of cells is not accompanied by severe depletion of ATP or by permeabilization of the plasma membrane to trypan blue. These results, together with known metabolic effects of nigericin, suggest that nigericin potentiates PDT by perturbing ion transport across either mitochondrial or plasma membranes.

Photodynamic therapy induces rapid cell death by apoptosis in L5178Y mouse lymphoma cells.

The mode of cell death of two strains of mouse lymphoma L5178Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmission electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

Basic radiobiological investigations of fast neutrons.

The radiobiological properties of a cyclotron-produced 43-MeV (p----Be) fast-neutron beam relative to gamma rays have been investigated using Chinese hamster V79 cells in culture. As expected, the relative biological effectiveness (RBE) of this neutron beam for cell killing was shown to increase as dose decreased, and the effectiveness per unit dose was slightly less compared to a 25-MeV (d----Be) neutron beam. By tracing single cells that formed microcolonies after irradiation, we found cell proliferation kinetics to be retarded to a greater extent by fast neutrons than by gamma irradiation. Following either neutron or gamma irradiation, a fraction of the irradiated cells failed to divide in the first postirradiation division and another fraction could produce as many as four generations of progeny before proliferation stopped. The properties of these cells presumed to be destined for death suggest that more than one mechanism and/or multistep process underlies the radiation-induced proliferative death. The fast-neutron beam was also found to be more effective quantitatively than gamma rays in producing DNA double-strand breaks (DSBs, measured by nondenaturing filter elution), and G1-phase chromosome fragments (measured by the premature chromosome condensation technique). However, the reverse was observed for DNA single-strand breaks (SSBs, measured by alkaline filter elution or hydroxylapatite uncoiling). Interestingly, both fast neutrons and gamma rays produced a large component of SSBs and DSBs with a fast-rejoining time constant of about 2-5 min, which appears to be independent of dose. The latter results could not resolve the possibility of lengthening the repair-time constant by increasing radiation dose within the range that is reflected by the shoulder of the survival curve, and consequently did not support the idea of repair saturation as a mechanism for the presence of the shoulder. The RBE for the hypoxanthine phosphoribosyl transferase mutation frequency per survivor at the 10% survival level was estimated to be 2.5, a value that is comparable to the RBE (2.1) for cell killing at the same survival level. Although most of the above-mentioned findings are compatible qualitatively with the relatively high-LET (linear energy transfer) nature associated with the fast-neutron beam, the significance of the action attributable to the mixture of LET could not be delineated in these experiments. Further, the biological significance of DSBs and chromosome aberration and the molecular mechanisms responsible for the repair and expression of these damaging processes remain to be elucidated.

Recurrence patterns in a prospective study of patients with stage II breast cancer treated with endocrine-chemotherapy.

Local-regional versus distant recurrence patterns were investigated for 311 patients with stage II node-positive breast cancer who were part of an endocrine-chemotherapy adjuvant breast cancer trial. After mastectomy patients were randomized to receive either cytoxan, methotrexate, and 5-fluorouracil (CMF) (1 year) or CMF with tamoxifen (1 year) with or without bacillus Calmette-Guérin (BCG). With a median follow-up of 92.1 months, 55.3% of the patients had recurrences. The first site of recurrence was local-regional for 31.4% of patients and distant for 68.6%. This pattern of first recurrence was not associated with treatment groups, menopausal status, race, estrogen receptor value, number of positive lymph nodes, or tumour diameter. Although patients with a first local-regional recurrence had a better overall prognosis compared with those with a first distant recurrence, 52.2% of those patients with an initial local-regional recurrence developed a distant recurrence within 12 months. Among patients who had a recurrence, 48.3% had a local-regional recurrence at some time during their follow-up. Conclusions from this study are (1) patterns of recurrence were not affected by the addition of antiestrogen therapy to chemotherapy; (2) for the variables tested, including number of positive nodes and tumor diameter, no association with recurrence patterns was found; and (3) most patients (52.2%) with a first local-regional recurrence will develop a distant recurrence within 1 year.

Results of conservative operations for breast cancer.

The results of conservative operations for breast cancer in 1,593 patients treated at the Cleveland Clinic between 1957 through 1975 are reported. During this period, we individualized our treatment of breast cancer depending on tumor size, location in the breast, and clinical stage of the disease. The following three principal operations were performed: modified radical mastectomy in 592 patients (37%), simple (total) mastectomy in 442 patients (28%), and partial (segmental) mastectomy in 291 patients (18%). Survival results at 5, 10, and 15 years are reported. Factors important in long-term survival included stage of the disease, number of lymph node metastases, delay in therapy, size of the tumor, histologic type, and estrogen receptor status; type of operation was not a significant factor. In this series, partial (segmental) mastectomy without radiation therapy provided five- to 15-year survival rates equal to modified radical mastectomy and simple (total) mastectomy.

Local-regional breast cancer recurrence following mastectomy.

Local-regional recurrence patterns were investigated in 1392 patients with breast cancer. Primary treatment for all patients included a mastectomy. Nine hundred seventeen patients had negative nodes and did not receive systemic therapy. Four hundred seventy-five patients had node metastases and were randomized to receive different combinations of chemoendocrine therapy. Follow-up ranged between 5 and 16 years. Two hundred thirty (25.8%) node-negative patients have had recurrences, with the initial recurrence being local-regional in 9.2%. Two hundred forty-two (50.9%) node-positive patients have had recurrences, with the initial recurrence being local-regional in 17.1%. Larger tumors and more extensive node involvement were associated with more first local-regional recurrences. The relative percent of first local-regional recurrence among patients in whom cancer recurred was similar for node-negative and node-positive patients (35.4% and 33.5%, respectively). In 63.6% of patients in whom cancer recurred, first local-regional recurrence were distant. Larger tumors, more extensive node involvement, and a shorter disease-free interval after mastectomy were associated with more rapid appearance of distant recurrence among these patients.

Photofrin II photosensitization is mutagenic at the tk locus in mouse L5178Y cells.

Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AlPcCl). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AlPcCl. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AlPcCl may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.

Post-treatment interactions of photodynamic and radiation-induced cytotoxic lesions.

The interaction of chloroaluminum phthalocyanine-sensitized photodynamic treatment and gamma-irradiation was studied in confluent murine L929 fibroblasts. When the cells were given the combined treatments and immediately subcultured for determination of cell survival by colony formation, the data indicate independent actions of each modality. However, when subculture was delayed for 1 h, a substantial fraction of cells treated with a sub-lethal dose of PDT followed by 5 Gy gamma-radiation detached from the monolayer. Most of these detached cells were no longer clonogenic. The mode of photosensitized cell killing was found to be different from that of ionizing radiation-induced cell killing. Photosensitized cell killing was accompanied by morphological changes in the cells and extensive DNA degradation within one hour following the treatment. When chloroaluminum phthalocyanine pretreated cells were exposed to a sublethal fluence of light (6 kJ/m2) and a lethal dose of gamma-radiation (5 Gy), DNA degradation was enhanced, and about 20% of the cell population appeared to undergo the type of cell death typical of photodynamic treatment. Thus, although different initial lethal lesions are induced by photodynamic treatment and by ionizing radiation, interactions may occur during processing of the damage.

New phthalocyanine photosensitizers for photodynamic therapy.

Six new aluminum and silicon phthalocyanines have been synthesized and their photocytotoxicity toward V79 cells has been studied. The compounds that have been prepared are: A1PcOSi(CH3)2(CH2)3N(CH3)2, I; A1Pc-OSi(CH3)2(CH2)3N(CH3)3+I-, II; CH3SiPcOSi(CH3)2(CH2)3N(CH3)2, III; HOSiPcOSi(CH3)2(CH2)3N(CH3)2, IV; HOSiPcOSi(CH3)2(CH2)3N(CH3)3+I-, V; and SiPc[OSi(CH3)2(CH2)3N(CH3)3+I-]2, VI. Relative growth delay values for compounds I-VI and relative cytotoxicity values for compounds I, II, IV, V and VI have been determined. Compounds I and II have been shown to be comparable in photocytotoxicity to what is presumed to be A1PcOH.xH2O, and compound IV has been shown to have greater activity. The classes of compounds to which these six compounds belong appear to have potential for photodynamic therapy.

Protection by the fluoride ion against phthalocyanine-induced photodynamic killing of Chinese hamster cells.

When a dilute F- solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCl, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F-. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA-protein crosslinks caused by light and this sensitizer were also inhibited by F-. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA-protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3-N(CH3)2 were not inhibited by F-. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F-, and that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA-protein crosslink formation indicates that the effects of F- can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F-.

DNA lesions and DNA degradation in mouse lymphoma L5178Y cells after photodynamic treatment sensitized by chloroaluminum phthalocyanine.

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabeled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.

Metoclopramide enhances the effect of photodynamic therapy on xenografted human squamous cell carcinoma of the head and neck.

OBJECTIVE: Photodynamic therapy (PDT) is a promising new treatment modality for head and neck cancer that is based on the uptake of a systemically administered photosensitizer in tumor tissue and local illumination of the lesion by a high-intensity visible light source, typically a tunable argon-pumped dye laser. We developed a new photosensitizer named silicon phthalocyanine [SiPc(OH) OSi(CH3)2(CH2)3N(CH3)2, abbreviated as SiPc IV], which yields superior PDT responses in vitro and in vivo compared with other clinically used photosensitizers. However, tumor regrowth following SiPc IV-based PDT is still a therapeutic problem. The benzamide derivatives, for example, have been shown to enhance tumor ablation when used during radiotherapy and chemotherapy. Therefore, we used metoclopramide hydrochloride, a benzamide derivative, to evaluate its effects on PDT response. DESIGN: Intradermally injected human squamous cell carcinoma cells were grown to 40 to 80 mm3 in athymic nude mice and irradiated with 675-nm light (75 J/cm2, 75 mW/cm2) 24 hours after the intraperitoneal injection of SiPc IV (1.0 mg/kg). Metoclopramide hydrochloride (2 to 48 mg/kg) was injected intraperitoneally 1 hour before and 24 and 48 hours after irradiation. RESULTS: Tumors exposed to PDT alone showed 80% to 90% tumor regression with regrowth in most animals within 20 days. Tumors treated with metoclopramide hydrochloride (48 mg/kg) plus PDT demonstrated 100% tumor regression without regrowth up to the time of killing (150 days). No observable toxic effects were clinically apparent with the high doses of metoclopramide. CONCLUSIONS: Our results show that administering metoclopramide in combination with PDT may be a promising approach to the management of head and neck cancer.

Update on intracavitary radiation in the treatment of bladder tumors.

A 15-year experience with intracavitary radiation for the treatment of selected bladder tumors is presented. The age range of these patients was 39 to 91 years, with an average of 65 years. There were 38 men and 17 women with multiple stage A bladder tumors and carcinoma in situ. Some patients were poor risks for treatment by total extirpative surgery. A simplified technique using a 25 mg. radium capsule as a central source is described for administration of 4,000 to 5,000 rad to the surface of the bladder. Most patients tolerate the presence of the radium catheter with little difficulty. No morbidity and no mortality were reported. In this series more than 60 per cent of the patients benefited from the therapy, with no recurrence noted in some cases after up to 8 1/2 years of followup. Intracavitary radiation is used in few clinics. Use of this modality does not preclude or complicate the subsequent use of other conservative measures or radical therapy if required. It is an effective procedure for treatment of multiple superficial and noninvasive tumors, including primary or recurrent carcinoma in situ, that are uncontrolled by other conservative measures.

On cats and the territorial imperative.

Of all the medical innovationes of our era, computed tomography is undoubtedly one of the most fascinating, and many nonradiologists are seeking entry into the field. If CT is to remain a radiological specialty, we must act to ensure that our training and experience are adequate to meet this new challenge.

The advantages of subtotal thyroidectomy and suppression of TSH in the primary treatment of papillary carcinoma of the thyroid.

Patients between the ages of 6 and 45 years with distant metastases from papillary carcinoma of the thyroid can be treated as effectively by subtotal thyroidectomy and suppressive doses of thyroid hormone as by total thyroidectomy followed by treatment with iodine 131 (131I). Moreover, distant metastases can be treated by either 131I or suppression as effectively after they are apparent on x-ray as they can be when treated in a subclinical stage. Therefore, in patients younger than 45 years old it is rarely necessary to perform a total thyroidectomy or to do frequent postoperative scans. In patients older than 44 or younger than 7 who have distant metastases or extensive involvement of both lobes, total or almost total thyroidectomy is justified if it can be done with minimal morbidity. In patients of this age group whose tumors fail to respond to suppressive doses of thyroid, 131I should be used. In view of the importance of diagnostic related groups (DRG) to the economy of hospitals, we note that the cost of total thyroidectomy, ablation by 131I, and intermittent body scans is at least three times that of less radical procedures which, in conjunction with suppression by thyroid feeding, give the same survival with less morbidity.

Half and total body radiation for carcinoma of the prostate.

Despite recent advances in the treatment of stage D carcinoma of the prostate many patients become refractory to all therapeutic modalities. Progressive and incapacitating pain is one of the most difficult symptoms to manage. Ten patients with severely symptomatic metastatic adenocarcinoma of the prostate have been treated with either single or sequential doses of half body radiation using 800 rad delivered by a Linac 10 mV linear accelerator. There were 15 courses of half body radiation delivered and a good to excellent response was noted in 11 instances. Results often were immediate and the duration of the response was variable. Treatment was well tolerated with no fatal complications. Half and total body radiation appears to offer significant palliation and its use with other forms of therapy warrants further investigation.

Simultaneous treatment with cisplatin and radiation therapy for advanced solid tumors: a pilot study.

To study the toxicity and efficacy of simultaneous cisplatin chemotherapy and radiation therapy, 13 patients with metastatic solid tumors were treated with cisplatin on a weekly, outpatient basis during palliative radiation therapy. The dose of cisplatin ranged from 20 to 50 mg/m2 weekly, with most patients receiving 40 mg/m2. Radiation therapy was administered in a variety of dose-fraction schedules. Toxic effects were moderate and consisted of emesis (12 patients), transient elevation off BUN (three patients), myelosuppression (three patients), and radiation reactions (two patients). Twelve of 13 irradiated lesions (91%) responded with at least a 50% reduction in biperpendicular diameter. Four of the six patients with metastatic melanoma had complete regression of treated lesions; another melanoma patient had a partial response. Responses were also seen in patients with mesothelioma, bladder cancer, squamous cell carcinoma of the head and neck, and oat cell lung cancer. Only one responding patient has had disease progression in the treated field after 2+ to 7+ months of followup; two other patients have died of disseminated disease. Weekly cisplatin administration during radiotherapy deserves further evaluation, especially in the primary management of unresectable tumors that are responsive to cisplatin alone.