Glutathione S-transferase alpha 4 induction by activator protein 1 in colorectal cancer.

Glutathione S-transferase alpha 4 (GSTA4) is a phase II detoxifying enzyme that metabolizes electrophiles and carcinogens including 4-hydroxy-2-nonenal (4-HNE), an endogenous carcinogen that contributes to colorectal carcinogenesis. In this study, we investigated GSTA4 expression and regulation in murine primary colonic epithelial cells, microbiome-driven murine colitis and human carcinomas. Exposure of YAMC cells to 4-HNE induced Gsta4 expression. Using an inflammation-associated model of colorectal cancer (CRC), Gsta4 expression increased in vivo in colon macrophages and serum after 2 weeks of colonization of IL-10 deficient (Il10-/-) mice with Enterococcus faecalis. Increased expression was noted after 9 months of colonization in colon macrophages and epithelia in areas of inflammation. In human colon biopsies, immunohistochemistry showed no GSTA4 expression in normal epithelial cells, whereas GSTA4 was strongly expressed in the neoplastic epithelia of invasive carcinomas. For tubular adenomas, increased expression was primarily noted in stromal macrophages. Increased GSTA4 was confirmed in established human CRC cell lines and associated with 4-HNE-protein adducts in human colon adenomas and CRC. Next, we showed that 4-HNE induced activation of c-Jun and Nrf2, two components of the oncogenic transcription factor AP-1. AP-1 inhibitors and gene-specific small interfering RNAs partially suppressed GSTA4 expression. Co-immunoprecipitation confirmed interactions between c-Jun and Nrf2 supporting a role for AP-1 in regulating 4-HNE-induced GSTA4 expression. These findings demonstrate GSTA4 activation during 4-HNE-induced neoplastic transformation in colorectal carcinogenesis. GSTA4 is a potential surrogate biomarker for CRC screening and should provide novel approaches for chemoprevention.

Abscopal effect of low-LET γ-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response.

Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.

Curcumin-altered p53-response genes regulate radiosensitivity in p53-mutant Ewing's sarcoma cells.

AIM: Curcumin has been demonstrated to have antitumor effects including radiosensitization by modulating many molecular targets including p53. Herein, we investigated the radiosensitizing effect of curcumin in p53 mutant Ewing's sarcoma (ES) cells. MATERIALS AND METHODS: Cells exposed to radiation with or without curcumin were examined for transcriptional and translational levels of p53 downstream targets and its influence in regulated apoptosis, DNA fragmentation, cell survival and clonal expansion. RESULTS: Curcumin significantly caused radiation induced expression of p21 and Bax, and reduced BclXl, Mcl1 with only marginal Bcl2 modulation. As a positive control to the study, both transcriptional and translational levels of p53 remained unchanged after radiation with/without curcumin. Conversely, curcumin caused radiation-induced apoptosis and DNA fragmentation. Consistently, curcumin enhanced radiation-induced cytotoxicity and clonal expansion. CONCLUSION: These results suggest that curcumin potentially radiosensitizes p53-mutant ES cells by regulating IR-modulated p53-response genes. However, the curcumin-associated p53-independent regulation of downstream targets remains to be explored.

Induced telomerase activity in primary aortic endothelial cells by low-LET gamma-radiation is mediated through NF-kappaB activation.

Our objective was to understand the mechanism through which cells that initially survive irradiation could acquire survival advantage. In this study, we show evidence that low-linear energy transfer gamma-radiation can induce telomerase enzyme activity in primary aortic endothelial cells, and that an upstream regulator, nuclear factor kappa B (NF-kappaB), controls this activation. Telomeric repeat amplification protocol (TRAP) assay showed that cells exposed to a dose of 2 Gy induce telomerase activity. Subsequent analysis revealed that radiation-induced telomeric activity is regulated at the transcriptional level by triggering activation of the promoter of the telomerase catalytic subunit, telomerase reverse transcriptase (TERT). A mechanistic study revealed that NF-kappaB becomes functionally activated upon radiation exposure and mediates the upregulation of telomerase activity by binding to the kappaB-binding region in the promoter region of the TERT gene. More significantly, elimination of the NF-small ka, CyrillicB recognition site on the telomerase promoter or inhibition of NF-small ka, CyrillicB by ectopically expressing the inhibitor protein IkappaBalpha mutant (Ismall ka, CyrillicBalpha(S32A/S36A))) compromises radiation-induced telomerase promoter activation. Consistent with the notion that NF-kappaB mediates gamma-ray-induced telomerase responses, TRAP assay revealed that ectopically expressed IkappaBalpha(S32A/S36A)) also attenuated telomerase enzyme activity. These findings indicate that NF-kappaB activation following ionizing radiation exposure may elicit a survival advantage by upregulating and maintaining telomerase activity.

Intensity-modulated radiosurgery for childhood arteriovenous malformations.

PURPOSE: Presentation of intensity-modulated radiosurgery (IMRS) for the treatment of inoperable, complex shaped pediatric arterio-venous malformations AVM. METHOD: Between 03/99 and 11/04, IMRS was delivered to seven children aged six to 18 years. Prescribed minimum doses ranged from 17.5 to 20 Gy (median 18 Gy). Radiosurgery planning and delivery used a serial tomotherapeutic IMRT technique (Peacock IMRT, North American Scientific/Nomos, Cranberry Township, PA) over two to four couch angles. A linear accelerator attached binary multi-leaf collimator was used to generate pencil beams of 10 mm by either 8.5 or 4.0 mm. Treatment planning employed an inverse treatment planning optimization algorithm. Parameters submitted to the treatment planning system were: prescription dose (PD), volume of target allowed to receive less dose (standard 3%), minimum dose (0.5 Gy less than PD), and maximum dose (200% of PD). Planning system specific IMRS target and tissue types were selected to prioritize dose conformality over dose homogeneity. The prescription isodose encompassed at least 95% of the target volume. We calculated conformality (CI) and homogeneity indices (HI) to characterize the quality of IMRS plans, and summarized preliminary clinical outcomes. FINDINGS: Target volumes ranged from 0.71 to 63.02 cm(3) (median 13.8 cm(3), 6/7 AVM larger than 10 cm(3)). Median CI was 1.07 (range 1.05 to 1.7) according to RTOG criteria. Median HI was 1.12 (range 1.09 to 1.23). During limited follow-up (median 32 months, range 5 to 53 months), two AVM completely obliterated at 19 and 22 months, and partial obliteration (>75%) was observed in three cases. No treatment-related side effects, other than acute nausea and temporary headaches interpreted as being associated with changes in cerebral blood distribution, were observed. CONCLUSIONS: IMRS can allow for highly conformal planning and delivery of radiosurgery radiation doses even if pediatric AVM target volumes are large and/or highly complex in shape. This technique has been seen to result in favorable preliminary outcomes, thus supporting future exploration of this technique in pediatric and adult patients.

Treatment of recurrent Merkel cell carcinoma: an analysis of 46 cases.

This report describes the course of recurrent Merkel cell carcinoma and defines possible treatment strategies for recurrent disease as seen in a long-term multisite retrospective analysis. Merkel cell carcinoma is a highly aggressive neuroendocrine skin cancer. Surgery and radiation therapy have been demonstrated ability to control this disease; however, recurrence is common. Systemic chemotherapy has, as yet, no presently defined role in primary treatment, and few conclusions can be reached regarding optimal treatment of disease recurrence. Forty-six patients were identified over the last 15 years in a retrospective analysis of patient records from several hospitals in the San Antonio, TX area. Hospital charts as well as outpatient treatment records were reviewed. Almost all patients developing recurrent disease did so within the first 2 years after primary treatment. Patients presenting distant disease had a median survival of 12 months, faring worse than those who display local or nodal disease. For patients with nodal or local recurrence, the mean survival after combination therapy (chemotherapy, radiation +/- surgery) was 36.5 months as compared with 17.5 months for those treated with a single modality (surgery or radiation or chemotherapy). The overall survival rate for the 46 patients with recurrence was 37%. Multimodality therapy has shown the best results for recurrent Merkel cell carcinoma thus far, and should be used if tolerated by the patient. Aggressive salvage surgery for local or nodal recurrence is encouraged, because this disease has a tendency to become more destructive upon recurrence. Adjuvant radiation therapy should also be used, if the patient has not exceeded their dose limitations. Disseminated disease, whether primary or recurrent, warrants further investigation in terms of optimal treatment.

Post-translational modification of I-kappa B alpha activates NF-kappa B in human monocytes exposed to 56Fe ions.

The objective of this study was to investigate whether heavy ion (56Fe) radiation exposure activates one of the key transcriptional regulators, nuclear factor-kappa B (NF-kappa B), in normal human monocytes (Mono Mac 6 cells: MM6). The study revealed that the exposure of MM6 cells to 56Fe ions resulted in increased NF-kappa B DNA-binding activity. The activation was both dose- and time-dependent, with a maximum response at the 2 h time point after a 0.7 Gy dose. Cells pre-incubated with inhibitors of the phosphorylation and proteasome signaling pathway, completely blocked heavy ion-induced activation of NF-kappa B. These results clearly indicate that 56Fe ions can induce NF-kappa B DNA-binding activity in normal human monocytes, that the activation is rapid and persistent, and that the heavy ion-induced activation of NF-kappa B is mediated through phosphorylation of I-kappa B alpha and the subsequent proteasome-dependent degradation pathway. Since activation of NF-kappa B by extracellular stimuli is implicated in inflammation, infection and cancer induction, as well as in protection of cells against insult, it will be important in subsequent studies to elucidate whether heavy ion-induced NF-kappa B activation is involved in downstream gene expression.

Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis.

Understanding the functional significance of the coordinate expression of specific corepressors and DNA-binding transcription factors remains a critical question in mammalian development. During the development of the pituitary gland, two highly related paired-like homeodomain factors, a repressor, Hesx1/Rpx and an activator, Prop-1, are expressed in sequential, overlapping temporal patterns. Here we show that while the repressive actions of Hesx1/Rpx may be required for initial pituitary organ commitment, progression beyond the appearance of the first pituitary (POMC) lineage requires both loss of Hesx1 expression and the actions of Prop-1. Although Hesx1 recruits both the Groucho-related corepressor TLE1 and the N-CoR/Sin3/HDAC complex on distinct domains, the repressor functions of Hesx1 in vivo prove to require the specific recruitment of TLE1, which exhibits a spatial and temporal pattern of coexpression during pituitary organogenesis. Furthermore, Hesx1-mediated repression coordinates a negative feedback loop with FGF8/FGF10 signaling in the ventral diencephalon, required to prevent induction of multiple pituitary glands from oral ectoderm. Our data suggest that the opposing actions of two structurally-related DNA-binding paired-like homeodomain transcription factors, binding to similar cognate elements, coordinate pituitary organogenesis by reciprocally repressing and activating target genes in a temporally specific fashion, on the basis of the actions of a critical, coexpressed TLE corepressor.

Effect of melatonin on cell growth, metabolic activity, and cell cycle distribution.

We have recently demonstrated that the pineal secretory product melatonin inhibits the key transcriptional regulator nuclear factor-kappa B (NF-kappa B). As the activation of NF-kappa B is known to regulate the expression of cellular genes associated with cell cycle progression, cell growth, and differentiation, we investigated the effect of melatonin treatment on several cellular processes. These include cell viability, metabolic activity, and cell cycle phase distribution. Human embryonic kidney (293S) cells were treated with melatonin at concentrations of 0.02, 0.2, or 2 mM. When cell viability was measured 24, 48, and 72 hr after continuous exposure to melatonin using the trypan blue dye exclusion method, no significant cell death was observed. Even after exposure to 2 mM melatonin for 72 hr, cell viability remained at 98%. In contrast, another antioxidant compound, pyrrolidine dithiocarbomate (PDTC), at a 2 mM concentration reduced cell viability to 80.7+/-2.1% as early as 24 hr compared with untreated controls (P<0.05). When the metabolic activity was determined at 24, 48, and 72 hr using the colorimetric MTT assay, no significant changes in metabolic activity were observed. Even if the cells were treated with 10 mM melatonin for 72 hr, the metabolic activity was similar to that of the control cells. When cell cycle analysis was performed by flow cytometry, no marked difference in cell cycle distribution was observed. Melatonin at a concentration of 2 mM, however, did slightly alter the cell cycle (percentage of S phase cells) at 48 hr. This study revealed that when 293S cells are treated with concentrations of melatonin up to 2 mM, no significant alterations in three important cellular functions occurred. Exogenously added melatonin appeared to have a limited influence on the normal functioning of the cells even when the exposure continued for 72 hr.

Tumor angiogenesis of low-grade astrocytomas measured by dynamic susceptibility contrast-enhanced MRI (DSC-MRI) is predictive of local tumor control after radiation therapy.

PURPOSE: To assess regional cerebral blood volume (rCBV) as a surrogate marker of angiogenesis in patients with low-grade fibrillary astrocytoma before radiation therapy and to correlate measured values with clinical outcome after fractionated stereotactic radiotherapy (FSRT). METHODS: Twenty-five patients with histologically proven fibrillary astrocytomas were examined using dynamic susceptibility contrast-enhanced MRI before radiotherapy. Radiotherapy was delivered to mean and median total doses of 60.9 and 60 Gy, respectively (range 55.8-66 Gy). During MRI for treatment planning, 55 T2*-weighted gradient echo images were acquired before, during, and after i.v. contrast-bolus injection. The acquired signal-time curves were converted into concentration-time curves. By normalization to an arterial input function, absolute and relative rCBV values were calculated. Measured pretherapeutic rCBV data were correlated to outcome in terms of local control after FSRT. RESULTS: Mean pretherapeutic rCBV for astrocytomas was 6.5 +/- 3.7 ml/100 g tissue. Mean and median follow-up times were 47.8 and 52 months, respectively. Fifteen tumors recurred during the period, with a mean and median latency of 39.1 and 42 months, respectively. Tumors recurring earlier than 42 months after FSRT showed a higher pretreatment rCBV than tumors recurring later and tumors in continued local control (8.12 +/- 4.48 ml/100 g vs. 6.0 +/- 2.3 ml/100 g and 4.73 +/- 2.47 ml/100 g; p = 0.02 and p = 0.03). The respective ratios of tumor rCBV in early recurrent tumors to gray matter and white matter rCBV were 0.98 +/- 0.38 and 2.17 +/- 1.36 as compared with 0.79 +/- 0.14 and 1.44 +/- 0.29 in locally controlled tumors (p = 0.074 and p = 0.056). CONCLUSIONS: In fibrillary low-grade astrocytomas, a noninvasive assessment of angiogenesis as indicated by rCBV measurement was feasible. The present data suggest that high pretherapeutic angiogenic activity in low-grade astrocytomas indicates a subgroup of tumors at higher risk for early local recurrence or malignant transformation after FSRT.

The TALON removable head frame system for stereotactic radiosurgery/radiotherapy: measurement of the repositioning accuracy.

PURPOSE: To present the TALON removable head frame system as an immobilization device for single-fraction intensity-modulated stereotactic radiosurgery (IMRS) and fractionated stereotactic intensity-modulated radiotherapy (FS-IMRT); and to evaluate the repositioning accuracy by measurement of anatomic landmark coordinates in repeated computed tomography (CT) examinations. METHODS AND MATERIALS: Nine patients treated by fractionated stereotactic intensity-modulated radiotherapy underwent repeated CTs during their treatment courses. We evaluated anatomic landmark coordinates in a total of 26 repeat CT data sets and respective x, y, and z shifts relative to their positions in the nine treatment-planning reference CTs. An iterative optimization algorithm was employed using a root mean square scoring function to determine the best-fit orientation of subsequent sets of anatomic landmark measurements relative to the original image set. This allowed for the calculation of the x, y, and z components of translation of the target isocenter for each repeat CT. In addition to absolute target isocenter translation, the magnitude (sum vector) of isocenter motion and the patient/target rotation about the three principal axes were calculated. RESULTS: Anatomic landmark analysis over a treatment course of 6 weeks revealed a mean target isocenter translation of 0.95 +/- 0.55, 0.58 +/- 0.46, and 0.51 +/- 0.38 mm in x, y, and z directions, respectively. The mean magnitude of isocenter translation was 1.38 +/- 0.48 mm. The 95% confidence interval ([CI], mean translation plus two standard deviations) for repeated isocenter setup accuracy over the 6-week period was 2.34 mm. Average rotations about the x, y, and z axes were 0.41 +/- 0.36, 0.29 +/- 0.25, and 0.18 +/- 0.15 degrees, respectively. Analysis of the accuracy of the first repeated setup control, representative of single-fraction stereotactic radiosurgery situations, resulted in a mean target isocenter translation in the x, y, and z directions of 0.52 +/- 0.38, 0.56 +/- 0.30, and 0.46 +/- 0.25 mm, respectively. The mean magnitude of isocenter translation was 0.99 +/- 0.28 mm. The 95% confidence interval for these radiosurgery situations was 1.55 mm. Average rotations at first repeated setup control about the x, y, and z axes were 0.24 +/- 0.19, 0.19 +/- 0.17, and 0.19 +/- 0.12 degrees, respectively. CONCLUSION: The TALON relocatable head frame was seen to be well suited for immobilization and repositioning of single-fraction stereotactic radiosurgery treatments. Because of its unique removable design, the system was also seen to provide excellent repeat immobilization and alignment for fractionated stereotactic applications. The exceptional accuracy for the single-fraction stereotactic radiosurgical application of the system was seen to deteriorate only slightly over a 6-week fractionated stereotactic treatment course.

An oblique arc capable patient positioning system for sequential tomotherapy.

A new patient positioning system has been designed and manufactured, allowing for the accurate delivery of obliquely oriented intensity modulated treatment arcs via a commercially available IMRT system. The ability to deliver such obliquely oriented intensity modulated arcs allows the commercial system to more closely approach a 4pi pencil beam delivery geometry which, in turn, allows for significant improvements in conformality for many tumor geometries. While the IMRT system delivered to this institution in the fall of 1996 was capable of planning for nonparallel plane delivery schemes, it proved incapable of delivering such treatments with acceptable accuracy. Because our early clinical experience revealed that certain patients could benefit significantly from such a delivery scheme we endeavored to design and manufacture an alternative treatment couch/patient positioning system (Xlator) which could overcome the limitations of the vendor supplied system. We present our initial evidence for the benefits of obliquely oriented intensity modulated treatment arcs, along with data demonstrating the inability of the original vendor supplied system to deliver such treatments with acceptable accuracy. The design of our new system is presented, as well as data demonstrating its ability to accurately deliver obliquely oriented intensity-modulated arcs. A detailed comparison of the performance of the Xlator and the vendor-supplied system is presented with regard to match line repeatability and hysteresis. Finally, the ability of the Xlator to deliver multiple couch angle sequential tomotherapy with spatial accuracy necessary to radiosurgical applications is demonstrated via a AAPM Report 54,TG-42 hidden target test. Readers note: The Xlator patient positioning system designed and patented here has recently come to be commercially available, and is currently marketed by the vendor under the name Crane II.

Antioxidant compounds interfere with the 3.

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner. Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product. These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments.

Targeting apoptosis by hydroxymethylacylfulvene in combination with gamma radiation in prostate tumor cells.

Hydroxymethylacylfulvene (HMAF) is a novel agent with alkylating activity and is a potent inducer of apoptosis that is currently undergoing Phase II clinical trials for prostate cancer. This study explored the pro-apoptosis and anti-proliferative potential of HMAF in combination with gamma radiation in human prostate tumor cell lines. Apoptosis was assessed based on the generation of fragmented DNA, a terminal transferase flow cytometry assay, and cell morphology. In each of the tumor cell lines examined, radiation alone induced a marginal level of apoptosis, even after a prolonged 48-h incubation after exposure. In contrast, HMAF alone was a potent inducer of apoptosis in prostate tumor cells but not in normal cells. Marked levels of apoptosis in tumor cells were also observed for the combination of HMAF with gamma radiation. When drug treatment preceded irradiation, at least additive levels of apoptosis were observed in both androgen-responsive and androgen-independent cells. The combined treatment with ionizing radiation and HMAF reduced the radiation dose needed for the same level of clonogenic survival up to 2.5-fold. The potentiation of apoptosis and reduction in the clonogenic survival of tumor cells occurred at HMAF concentrations lower than that which reduced survival to 10% and at doses up to 6 Gy. No potentiation of apoptosis or clonogenic inhibition was noted in normal cells. These results suggest that the combination of HMAF with gamma radiation may have clinical utility for treatments of prostate cancer.

Prognostic value of p53 for local failure in mastectomy-treated breast cancer patients.

PURPOSE: The loss of p53 function is a recognized adverse prognostic factor in invasive breast cancer. Several studies have shown a relationship between the nuclear accumulation of p53 protein (a surrogate marker of p53 inactivation) and poor disease-free and overall survival. In general, however, these studies did not report the prognostic value of p53 for local failure, which we have therefore assessed retrospectively here. MATERIALS AND METHODS: Accumulation of p53 protein was evaluated by immunohistochemistry in 1,530 mastectomy-treated breast cancer patients (259 radiation therapy [RT]- and 1,271 mastectomy only [No RT]-treated patients). Statistical comparisons were made between p53 protein accumulation, estrogen/progesterone receptors, nodal status, tumor size, and local failure rate (LFR). Local failure was defined as tumor recurrence involving the chest wall and/or the ipsilateral supraclavicular/axillary lymph nodes. The median follow-up period was 62 months. RESULTS: In the No RT group, the LFR was 9.1% and 16. 5% in p53-negative and p53-positive patients, respectively (P<.001). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (relative risk [RR], 1.7; 95% confidence interval [CI], 1.2 to 2.4). Nodal status and tumor size were also significant factors. In the RT group, the LFR was 9.3% and 21.5% in p53-negative and p53-positive patients, respectively (P = .009). Multivariate analysis revealed that p53 protein accumulation was significantly associated with an increased risk of local relapse (RR, 2.5; 95% CI, 1.1 to 5.7), as was nodal status. CONCLUSION: Nuclear accumulation of p53 protein is independently associated with a significantly increased local failure rate in breast cancer patients treated with mastectomy, with or without radiation.

Melatonin and protection from genetic damage in blood and bone marrow: whole-body irradiation studies in mice.

The objective of this study was to examine the potential radioprotective properties of pharmacological doses of melatonin in whole-body irradiated mice. CD2-F1 male mice were treated with melatonin, a secretory product of the pineal gland, and then whole-body irradiated with an acute dose (150 cGy) of 137Cs gamma rays. Peripheral blood and bone marrow cells were examined for genetic damage, which was determined by comparing the incidence of micronuclei (MN) in both melatonin pre-treated and non-treated irradiated animals (and control mice). The percentages of polychromatic erythrocytes (PCEs) in unirradiated mice ranged between 3.1 +/- 0.23 and 3.2 +/- 0.19 in the peripheral blood and between 51.0 +/- 2.03 and 52.8 +/- 2.00 in the bone marrow. Whole-body irradiation resulted in a significant decrease in the percentages of PCEs in the peripheral blood and bone marrow cells. In both tissues, irradiated mice that were pre-treated with melatonin (5 or 10 mg/kg) exhibited a dose-dependent increase in the observed incidence of PCEs relative to the expected incidence. The incidence of MN in unirradiated mice ranged between 4.2 +/- 0.92 and 4.6 +/- 0.97 in the peripheral blood and between 5.0 +/- 1.05 and 5.5 +/- 1.08 in the bone marrow. Whole-body irradiation resulted in a significant increase in the incidence of MN in both tissues. In both tissues, irradiated mice that were pre-treated with melatonin exhibited a significant and dose-dependent reduction in the observed incidence of MN (relative to the expected incidence). Under the experimental conditions tested, the data indicate that melatonin has the ability to protect the genetic material of hematopoietic cells of mice from the damaging effects of acute whole-body irradiation.

Selective cytotoxicity of 3-amino-L-tyrosine correlates with peroxidase activity.

In the presence of 3-amino-L-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10(6) cells versus 26.1 and 5.0 ng/10(6) cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and > 70% reduction in cell viability after 4 d of culture in the presence of 100 microg 3-AT per ml. Higher concentrations of 3-AT (up to 400 microg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1-3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 microg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 microg 3-AT per ml, but concentrations less than 400 microg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 microg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 microg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated "aminomelanin" formation is hypothesized.

Interaction of hyperthermia with Taxol in human MCF-7 breast adenocarcinoma cells.

Hyperthermia treatments (43 degrees C, 1 h) were performed on exponentially growing MCF-7 breast adenocarcinoma cells at the beginning, middle, or end of 24 h incubations of the cells in vitro with Taxol (paclitaxel). When the cells were heated at the beginning or middle of the Taxol incubation, the hyperthermia treatment protected against the toxic effect of each of the Taxol concentrations examined (5, 10 and 100 nM). Consistent with earlier studies, Taxol treatment at 37 degrees C resulted in an accumulation of greater than 94% of the cells in G2/M at 24 h. Heating the cells at the middle or end of the Taxol treatment resulted in a similar accumulation. However, heat treatment during the first hour of Taxol exposure resulted in a significantly smaller percentage of cells (approximately 50%) in G2/M. HPLC analysis showed that at 37 degrees C, Taxol uptake into MCF-7 cells approached maximum within 0.25 h and increased only slightly more over the next 11.75 h. The parental Taxol level was markedly lower by 24 h. In contrast, 1 h hyperthermia treatments at the beginning or middle of the Taxol incubation resulted in higher Taxol concentrations at 12 and 24h, and higher intracellular concentrations overall than at 37 degrees C. These results indicate that hyperthermia inhibits Taxol related cell cycle effects and cytotoxicity, in spite of causing higher concentrations of Taxol to be present in heated cells.

Melatonin and protection from whole-body irradiation: survival studies in mice.

The radioprotective ability of melatonin was investigated in mice exposed to an acute whole-body gamma radiation dose of 815 cGy (estimated LD50/30 dose). The animals were observed for mortality over a period of 30 days following irradiation. The results indicated 100% survival for unirradiated and untreated control mice, and for mice treated with melatonin or solvent alone. Forty-five percent of mice exposed to 815 cGy radiation alone, and 50% of mice pretreated with solvent and irradiated with 815 cGy were alive at the end of 30 days. Irradiated mice which were pretreated with 125 mg/kg melatonin exhibited a slight increase in their survival (60%) (p=0.3421). In contrast, 85% of irradiated mice which were pretreated with 250 mg/kg melatonin were alive at the end of 30 days (p=0.0080). These results indicate that melatonin (at a dose as high as 250 mg/kg) is non-toxic, and that high doses of melatonin are effective in protecting mice from lethal effects of acute whole-body irradiation.

Melatonin: possible mechanisms involved in its 'radioprotective' effect.

Peripheral blood samples were collected from four human volunteers before, and at 1 and 2 h after a single oral dose of 300 mg of melatonin. From each sample, separate aliquots of whole blood were exposed to 100-150 cGy gamma radiation in vitro to determine the extent of genetic damage. Irradiated lymphocytes from all volunteers which were collected after melatonin ingestion exhibited a significantly decreased extent of primary DNA damage and reduced frequencies of chromosomal aberrations and micronuclei, as compared with similarly irradiated cells collected before the oral dose of melatonin. The extent of the melatonin-associated decrease in primary DNA damage was less than the corresponding decrease observed in the incidence of chromosomal aberrations and micronuclei; the latter assays required an additional post-irradiation incubation of the cells at 37+/-1 degreesC for 48 and 72 h, respectively. The possible mechanisms involved in the radioprotective influence of melatonin are discussed.