Helene Z. Hill

Free radical-initiated and gap junction-mediated bystander effect due to nonuniform distribution of incorporated radioactivity in a three-dimensional tissue culture model

Abstract Bishayee, A., Hill, H. Z., Stein, D., Rao, D. V. and Howell, R. W. Free Radical-Initiated and Gap Junction-Mediated Bystander Effect due to Nonuniform Distribution of Incorporated Radioactivity in a Three-Dimensional Tissue Culture Model. To investigate the biological effects of nonuniform distribution of radioactivity in mammalian cells, we have developed a novel three-dimensional tissue culture model. Chinese hamster V79 cells were labeled with tritiated thymidine and mixed with unlabeled cells, and multicellular clusters (∼1.6 mm in diameter) were formed by gentle centrifugation. The short-range β particles emitted by 3H impart only self-irradiation of labeled cells without significant cross-irradiation of unlabeled bystander cells. The clusters were assembled in the absence or presence of 10% dimethyl sulfoxide (DMSO) and/or 100 μM lindane. DMSO is a hydroxyl radical scavenger, whereas lindane is an inhibitor of gap junctional intercellular communication. The clusters were maintained at 10.5°C for 72 h to allow 3H decays to accumulate and then dismantled, and the cells were plated for colony formation. When 100% of the cells were labeled, the surviving fraction was exponentially dependent on the mean level of radioactivity per labeled cell. A two-component exponential response was observed when either 50 or 10% of the cells were labeled. Though both DMSO and lindane significantly protected the unlabeled or bystander cells when 50 or 10% of the cells were labeled, the effect of lindane was greater than that of DMSO. In both cases, the combined treatment (DMSO + lindane) elicited maximum protection of the bystander cells. These results suggest that the bystander effects caused by nonuniform distributions of radioactivity are affected by the fraction of cells that are labeled. Furthermore, at least a part of these bystander effects are initiated by free radicals and are likely to be mediated by gap junctional intercellular communication.

Dimethyl triazeno imidazole carboxamide and combination therapy for melanoma IV. Late results after complete response to chemotherapy (central oncology group protocols 7130, 7131, and 7131A)

The results of three Phase III studies of DTIC in 580 patients with metastatic melanoma were reviewed to evaluate the subsequent course of 26 patients who achieved a complete response (CR) to chemotherapy. The majority (17 of 26) of these patients had soft tissue metastases. Six of the 26 patients remained in CR at last report (30–259 weeks), two died of other causes while remaining free of melanoma, and 18 relapsed and died. Ninety‐five percent of the 26 patients were alive at 1 year, and survival was 31.1% at 72 months. Seven of the eight patients with sustained remission received chemotherapy for at least 6 months after CR developed, whereas 10 of 18 relapsing patients were treated for less than 6 months after CR was achieved. Long‐term sustained CR to chemotherapy occurs in 1% to 2% of patients treated with DTIC, and late relapse is rare in patients who remain in CR for 2 years. Cancer 53:1299‐1305, 1984.

Induced melanin reduces mutations and cell killing in mouse melanoma.

When melanin absorbs light energy, it can produce potentially damaging active oxygen species. There is little doubt that constitutive pigment in dark-skinned individuals is photoprotective against skin cancer, but induced pigment-as in tanning-may not be. The first step in cancer induction is mutation in DNA. The most suitable systems for evaluating the role of melanin are those in which pigment can be varied and mutations can be measured. Several cell lines from Cloudman S91 mouse melanoma can be induced to form large quantities of melanin pigment after treatment with a number of different agents enabling comparison of mutant yields in the same cells differing principally in pigment concentration. In these studies, melanin was induced with synthetic alpha-melanocyte-stimulating hormone and with isobutyl methyl xanthine in the cell line S91/mel. The former inducer produced about 50% more pigment than the latter. Survival and mutation induction at the Na+/K(+)-ATPase locus were studied using ethyl methane sulfonate (EMS), a standard mutagen and five UV lamps emitting near monochromatic and polychromatic UV light in the three wave-length ranges of UV. There was greater protection against killing and mutation induction in the more heavily pigmented cells after exposure to EMS and after irradiation with monochromatic UVC and UVB. There was significant protection against killing by polychromatic UVB + UVA (FS20), but the small degree of protection against mutation was not significant. No significant change in killing and mutation using the same protocol was seen in S91/amel, a related cell line that does not respond to these inducers. No mutants were produced by either monochromatic or polychromatic UVA at doses that killed 50% of the cells. Our results show that induced pigment-shown earlier to be eumelanin (K. A. Cieszka et al., Exp. Dermatol. 4, 192-198, 1995)-is photo- and chemoprotective, but it is less effective in protection against mutagenesis by polychromatic UVB + UVA in a spectrum that more nearly approximates the solar spectrum.

Comparative Action Spectrum for Ultraviolet Light Killing of Mouse Melanocytes from Different Genetic Coat Color Backgrounds

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony‐forming ability after irradiation. The cell lines were wild‐type black (melan‐a) and the mutants brown (melan‐b) and albino (melan‐c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200–280 Dm), 82.3% output at 254 nm, TL01 (UVB, 280–320 nm), 64.2% at 310–311 nm, FS20, broadband with peak output at 312 nm and Alisun‐S (UVA, 320–400 nm), broadband with peak output at 350–354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild‐type melan‐a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan‐c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan‐b was more sensitive than melan‐a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6–4 photoproducts) produced in melan‐a than in melan‐c cells by UVC, UVB and FS20 lamps. Unlike melan‐c, melan‐a and melan‐b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin‐like centers. The contribution of pheome‐lanin was higher in melan‐b than in melan‐a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild‐type melan‐a melanocytes were well melanized and ellipsoidal, whereas those of melan‐b melanocytes tended to be spherical. In the albino line (melan‐c) the melanocytes contained only early‐stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.

Melanin Both Causes and Prevents Oxidative Base Damage in DNA: Quantification by Anti-Thymine Glycol Antibody

The present study employs immunological methods to measure modified bases in DNA. A polyclonal antibody specific for thymine glycol was used to quantify the level of thymine glycol in calf thymus DNA gamma-irradiated in solutions containing varying concentrations of DOPA-eumelanin. Melanin decreased the number of thymine glycols produced by 200 Gy at low melanin concentrations. At high melanin concentrations, the number of thymine glycols increased. Thymine glycol was also produced in unirradiated DNA-eumelanin mixtures. DOPA-eumelanin was found to produce single-strand breaks in supercoiled phi X174 RF DNA. The breaks were measured by conversion of form I to form II as detected by agarose gel electrophoresis. The level of damage produced by melanin could be modulated by agents known either to stabilize or to scavenge active oxygen species. These studies demonstrate that melanin can both scavenge and generate active free radicals.

Studies of Chloroplast Development in Euglena: XIII. Variation of Ultraviolet Sensitivity with Extent of Chloroplast Development

Ultraviolet (UV) inactivation of green colony-forming ability of several different types of Euglena gracilis var. bacillaris was studied. The observed target numbers are not widely different, while the doses required to produce a single inactivation event (D(o)) vary with the type of cell used. In dark-grown cells adapting to the light in resting medium and in an X-ray-induced mutant, D(o) is proportional to the chlorophyll content of the cells. However, in hyperdeveloped cells which contain abnormally high amounts of chlorophyll, the correlation does not hold, suggesting that it is not chlorophyll per se which is responsible for the differences observed. D(o)s of colony-forming ability (viability) of light-grown and dark-grown cells are found to differ by the same factor as those of green colony-forming ability. Stationary phase and exponential phase cells show a small difference in D(o) with no obvious difference in target multiplicity. The multiplicity of the various target curves has been re-evaluated by computer and found to be between 30 and 40.

COMPARATIVE ACTION SPECTRA FOR PYRIMIDINE DIMER FORMATION IN CLOUDMAN S91 MOUSE MELANOMA and EMT6 MOUSE MAMMARY CARCINOMA CELLS

Abstract— Pyrimidine dimer formation in melanotic mouse melanoma cells, Cloudman S91H‐. and iii mouse mammary carcinoma cells, EMT6, was compared as a function of wavelength by irradiating equal numbers of cells from the two cell lines simultaneously. More dimers were formed in EMT6 than in S91H– by light of wavelengths less than 289 nm, while light of higher wavelengths caused equivalent dimer formation, as measured by the Micrococcus luteus UV‐endonuclease assay. The cells of S91 flare lightly melanotic, yet shielding at lower wavelengths is considerable. It is speculated that melanin pigmentation arose by selection during an evolutionary period when UV‐C light reaching the earths surface was significantly greater than it is today.

Detection of inborn errors of metabolism

Human fibroblasts, cultured on glass microscope slides, were examined autoradiographically for their ability to incorporate radioactivity from Na‐propionate‐l‐ 14C into TCA‐insoluble cell material. Cells from patients with propionic acidemia (PA) and methylmalonic acidemia (MMA), both B12‐sensitive and B12‐insensitive, incorporate little or no radioactivity and thus can be readily distinguished from cells of a number of individuals with no defects in this pathway. Incorporation in cells from non‐MMA individuals is significantly enhanced in a basal medium by high levels of glucose. This procedure should be readily adaptable for use in screening for disorders of propionate metabolism, particularly before birth.

The Degree of Pigmentation Modulates the Radiosensitivity of Human Melanoma Cells

Abstract Kinnaert, E., Morandini, R., Simon, S., Hill, H. Z., Ghanem, G. and Van Houtte, P. The Degree of Pigmentation Modulates the Radiosensitivity of Human Melanoma Cells. The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.

Differential regulation of full-length genome and a single-stranded 7S DNA along the cell cycle in human mitochondria

Mammalian mitochondria contain full-length genome and a single-stranded 7S DNA. Although the copy number of mitochondrial DNA (mtDNA) varies depending on the cell type and also in response to diverse environmental stresses, our understanding of how mtDNA and 7S DNA are maintained and regulated is limited, partly due to lack of reliable in vitro assay systems that reflect the in vivo functionality of mitochondria. Here we report an in vitro assay system to measure synthesis of both mtDNA and 7S DNA under a controllable in vitro condition. With this assay system, we demonstrate that the replication capacity of mitochondria correlates with endogenous copy numbers of mtDNA and 7S DNA. Our study also shows that higher nucleotide concentrations increasingly promote 7S DNA synthesis but not mtDNA synthesis. Consistently, the mitochondrial capacity to synthesize 7S DNA but not mtDNA noticeably varied along the cell cycle, reaching its highest level in S phase. These findings suggest that syntheses of mtDNA and 7S DNA proceed independently and that the mitochondrial capacity to synthesize 7S DNA dynamically changes not only with cell-cycle progression but also in response to varying nucleotide concentrations.