Activity of cathepsin D and alpha(1)-antitrypsin in the blood serum of patients with mammary carcinoma.

UNLABELLED: THE AIM of this study was to determine the activity of cathepsin D and alpha(1)-antitrypsin in the blood serum of patients with mammary carcinoma. PATIENTS AND METHODS: The study was conducted on 52 women operated for a unilateral breast tumor, divided into two groups, according to the number of metastases and tumor size. Cathepsin D activity was determined using the method of Anson, while alpha(1)-antitrypsin activity was determined according to the Eriksson method. RESULTS: Both groups of patients with mammary carcinoma were found to have higher activity of cathepsin D before the treatment compared to healthy females. After the surgery the enzyme activity increased significantly, whereas 6 months after the surgery it generally decreased. The activity of alpha(1)-antitrypsin was significantly lower in patients before the treatment than in the controls, while after 6 months an increase in alpha(1)-antitrypsin activity was observed. The correlation between activity of cathepsin D and alpha(1)-antitrypsin was revealed. High enzyme activity and low alpha(1)-antitrypsin activity may result from the stage of neoplastic transformation. CONCLUSION: The determination of cathepsin D activity together with alpha(1)-antitrypsin activity may serve as useful biochemical marker in monitoring of malignant changes in breast tumor.

Activity of antioxidant enzymes and concentration of lipid peroxidation products in selected tissues of mice of different ages, both healthy and melanoma-bearing.

The activity of antioxidant enzymes and the concentration of the lipid peroxidation product malondialdehyde (MDA) as indicator of oxidative damage were determined in selected tissues of healthy mice and transplanted B16 melanoma-bearing mice with increasing age. A total of 60 male mice were divided into 6 groups. Groups 1, 2 and 3 consisted of tumor-free, healthy mice aged 1, 9 and 16 months, respectively (average life span: 2 years). Groups 4, 5 and 6 consisted of mice of the same age as the healthy mice, but given intraperitoneally 10(6) cells of B16 melanoma for 2 weeks. An increase in the concentration of MDA was found in all the studied tissues (brain, liver, lungs, erythrocytes) and blood plasma of 16-month old healthy mice compared with the younger ones. The activity of superoxide dismutase (SOD) and catalase (CAT) was elevated in the brain and the activity of CAT and glutathione peroxidase (GPx) in the liver of aged healthy mice. The transplantation of melanoma caused an increase of the concentration of MDA and of the activity of all studied enzymes in all tissues. This elevation was most pronounced in the youngest mice group 4 and was higher than in the oldest healthy group 3. Thus, these early changes of the "(anti-)oxidative status" in the investigated tissues caused by the tumor development have similarities with age-associated alterations of healthy mice, especially in regard to MDA in all tissues or SOD and CAT in brain.

Activity of antioxidant enzymes and concentrations of thiobarbituric acid reactive substances (TBARS) in melanotic and amelanotic Bomirski melanoma tissues in the golden hamster (Mesocricetus auratus, Waterhouse).

The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx), as well as the concentration of thiobarbituric acid reactive substances (TBARS) in tissues of transplantable melanoma in the golden hamster were measured and compared. Ten inbred male hamsters were used for the experiment. They were divided into two groups and were given Bomirski melanoma cells subcutaneously. The first group was given melanotic (Ma) melanoma cells. The second group was given amelanotic (Ab) melanoma cells. Thirty days after the transplantation the hamsters were dissected and the tumor tissues were taken and homogenized. A statistically significantly higher activity of the measured antioxidant enzymes was found in homogenates of Ma tumor than in homogenates of the Ab tumor. Activity of SOD is 8% higher in melanotic melanoma, 24% higher in CAT, and 45% higher in GSHPx. Statistically significant differences between TBARS concentrations were not confirmed. The higher activity of antioxidant enzymes in the melanotic tumor is a result of increased generation of oxygen-derived free radicals. It is presumed that it is strictly connected with intensified production of quinone and semiquinone radicals in the process of melanogenesis.

[Decreased stimulation of glycosaminoglycan synthesis by human skin fibroblasts by interleukin 6 within the scope of in vitro aging]. / Verminderte Stimulation der Glycosaminoglycansynthese von humanen Hautfibroblasten durch Interleukin-6 im Rahmen der in vitro Alterung.

Addition of human recombinant interleukin 6 (IL-6) to culture medium (supplemented MEM without or with 10% fetal calf serum (FCS)) of human skin fibroblasts exerted a stimulating effect in a dose-dependent manner on glycosaminoglycan (GAG) synthesis, including hyaluronic acid (hyaluronan) synthesis, of young (phase-II) skin fibroblasts in concentrations of 1 ng/ml and 10 ng/ml. Stimulation was mainly due to an increase in extracellular GAGs (secreted into culture medium), and to a lesser extent to an increase in peri- and intracellular GAGs. Stimulation with 1 ng/ml and 10 ng/ml IL-6 led to an increase in hyaluronic acid from 48% to 61% (-FCS) and from 77% to 90% (+10% FCS), respectively. Maximum stimulation, with and without FCS, was achieved by 10 ng/ml IL-6. Compared to young (phase-II) cells, senescent (phase-III) cells, showed no significant stimulation of total GAG (including hyaluronic acid) synthesis by IL-6. The diminished response of GAG- and hyaluronic acid synthesis during aging of these in vitro cultured fibroblasts should motivate further research if similar processes occur during aging in an organism.

Energetic stress induces premature aging of diploid human fibroblasts (Wi-38) in vitro.

Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na(+)/K(+)-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts.

Influence of quercetin on B16 melanotic melanoma growth in C57BL/6 mice and on activity of some acid hydrolases in melanoma tissue.

Quercetin (QC) (5, 7, 3', 4' -tetra oxyflavonolol) is an ubiquitous flavonoid in many plants. The influence of QC on the growth of B16 melanotic melanoma in C57BL/6 mice and activity of some acid hydrolases in the tumor homogenates were investigated. Two series of experiments were carried out: In the first experimental group mice were inoculated s.c. with 10(6) of tumor cells (TC) suspended in 1 ml of saline. TC were obtained from the current serial passages. In the second series of experimental group mice were inoculated with melanoma cells preincubated 15 min. in different concentrations of QC. Mice of both series were divided into three subgroups. Mice of the first series were treated with QC i.p. every second day in a dose of 0.1 mg, 0.5 mg or 1.0 mg (total dose of 1.0 mg, 5.0 mg or 10.0 mg per mice). Animals of the second series did not obtain any treatment. After the nineteenth day of experiment the mice were killed, tumors excised and weighed. Tumor tissue pieces were homogenized for enzyme activity determination. Fragments of tumor tissue were taken for electron microscopy (EM) investigation. In mice injected i.p. with QC mean tumor weight was significantly higher than in control I. The mean tumor weight in the first experimental group was higher than in control from 170% to 196% and in the second experimental group from 69% to 147%. Enzymes activity was also higher in both experimental groups as compared to controls. Arylsulphatase activity in the first group was higher from 102% to 144% and in the second one - from 97% to 115% than in control I. Acid phosphatase activity was higher from 100% to 155% in the first experimental group and from 56% to 161% in the second one. Cathepsin D activity was greater from 133% to 333% and from 113% to 300%, respectively. EM studies revealed the presence of greater number of Golgi structures and primary lysosomes in experimental groups of tumors (mice treated with QC and mice with melanoma preincubated in QC). These results clearly indicate that QC significantly enhances melanotic melanoma growth and increases acid phosphatase and cathepsin D activity in these tumors. The mechanism of QC action on the melanotic melanoma is not fully understood and remains to be defined.

Energetic stress in combination with tumor necrosis factor-alpha (TNF-alpha) induces apoptosis of human fibroblasts (WI-38) in vitro: reduced responsiveness of senescent cells.

The NaCl content of the culture medium of human fibroblasts (WI-38) was elevated from 0.12 M to 0.23 M. Previously it had been shown that exposure of fibroblasts to such high NaCl conditions resulted in a rise of both glycolysis and respiration ("energetic stress"), mainly due to increased demands of energy for active ion transport ("sodium pump"). While "young" (Phase II) and "senescent" (Phase III) cells did not show a significant increase of apoptosis over the basal rate (ca. 4%) under treatment with either a high-NaCl medium or TNF-alpha (10 nM) alone, combined treatment resulted in a strong increase in Phase II cells and a significantly lesser rise in the case of "senescent" Phase III cells. We conclude, therefore, that energetic stress stimulates sensitivity to apoptosis by (in the presence of) TNF-alpha, especially pronounced in potentially replicating "young" as compared with irreversible postmitotic ("senescent") fibroblasts. Possible causes of this differential responsiveness and implications for the in vivo situation (in the organism) were discussed.

The role of apoptosis in aging and age-related disease: update.

Programmed death of cells by apoptosis is regarded as a protective mechanism of the organism against an accumulation and spread of defective cells. The rate of apoptosis is elevated in most types of aging cell populations. However, there are also findings about a decreased susceptibility of senescent cells in vivo and in vitro, particularly to apoptosis induced by oxidative and energetic stress. Mitochondria appear to have a key function in apoptosis regulation. Thus, apoptosis can be induced by defective mitochondrial oxidative phosphorylation. The role of apoptosis in aging and age-related disease was outlined for different organs (brain, cardio-vascular system, immune system, intestine, macula of the eye, Langerhans islets, prostate gland, oocytes of ovaries). The age-related intensification of this dismantling system of cells seems to highlight the deterioration of tissue and organ structure and function in aging.

Microspectrofluorometry and fluorescence imaging in the study of human cytopathology.

The study of energy pools and dynamics of specific pathways in living cells by microspectrofluorometry and fluorescence imaging produces spectral and topographic images characterizing structural and functional changes associated with cytopathology. Microspectro-fluorometry and fluorescence imaging have been applied, together with organelle morphometry to a number of cells mimicking certain cytopathologies, including melanoma cells, long-term malignant cells, and gene-defective cells. These investigations of cellular pathology indicate that there is a convergence of various physiopathological processes. Cellular states that have similarities include senescence, detoxification, and transformation. While the NAD(P)H metabolic transients have been studied before, our emphasis in this article is on very rapidly scanned fluorescence images related to organelle integration and photoinduced cellular senescence.

[Decreased stimulation of glycosaminoglycan synthesis in cultured human skin fibroblasts within the scope of in vitro aging caused by interleukin 1]. / Verminderte Stimulation der Glycosaminoglycansynthese von kultivierten humanen Hautfibroblasten im Rahmen der In-vitro-Alterung durch Interleukin-1.

Addition of human recombinant interleukin-1 (IL-1 alpha or IL-1 beta) to culture medium (supplemented MEM without or with 1%, 10%, or 20% fetal calf serum (FCS)) of human skin fibroblasts exerted a stimulating effect in a dose-dependent manner on glycosaminoglycan (GAG) synthesis, including hyaluronic acid synthesis, of young (Phase II) skin fibroblasts in concentrations of 4, 20, or 100 pg/ml. Stimulation was due to increase in total GAGs, namely extracellular (secreted into culture medium) and cell-bound (pericellular) GAGs. Stimulation with 100 or 200 pg/ml IL-1 beta led to a relative increase (total GAGs: 100%) in hyaluronic acid from 49% to 64 (-FCS) and from 79% to 92% (+10% FCS), respectively. The increase in total GAG synthesis was mainly due to increased synthesis of hyaluronic acid. Maximum stimulation, with and without FCS, was reached by 100 pg/ml IL-1 beta. Compared to young (Phase II) cells senescent (Phase III) cells showed significantly diminished stimulation of hyaluronic acid synthesis by IL-1 beta. Both, IL-1 alpha and IL-1 beta, showed no influence on DNA synthesis. These results suggest that both, IL-1 beta and IL-1 alpha, stimulate GAG and especially hyaluronic acid synthesis, but not DNA synthesis, in vitro. The diminished response of GAG and hyaluronic acid synthesis during ageing of these in vitro cultured fibroblasts gives support to the hypothesis that similar processes might occur during ageing in organisms.

[Modulation of oxidative stresses in human aging skin]. / Modulation des oxidativen Stresses in der humanen Altershaut.

Oxidative stress (UV irradiation, free radicals) plays a significant role in aging. Coenzyme Q10 (CoQ10) and exogenously applied antioxidants can significantly reduce the formation of oxidative stress with increasing age. In our in vitro and in vivo experiments concerning the parameters of ultraweak photon emission (UPE), intracellular thiol status, mitochondrial membrane potential and cell vitality, we demonstrated a diminished resistance in keratinocytes of old donors against UV irradiation. This reduced epidermal resistance against oxidative stressors, i.e. UV irradiation, can be improved by topical application of CoQ10 and antioxidants like alpha-glucosylrutin (15). Furthermore, our in vivo investigations show that wrinkles around the region of the eyes ("crow feet") could be reduced by long-term application of CoQ10.

Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology.

The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell-membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors. Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy. In the multiprobe-multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations. 'Smart' instrumentation does not mean 'smart' probes. It turns out that the cell's own endogenous probes, the fluorescent coenzymes, may be akin to 'smart' probes, open to study in situ of many-fold interrelated pathways in cell energetics and dynamics. Resolution at the micro- and even nano-compartment levels is not altogether impossible. Thus an innovative search in terms of what may be called 'intracellular reconnaissance with fluorescent probes and biopharmaceuticals' necessitates recourse to multiple tentative probings along the pleiotropic mechanisms as far in resolution as one can go. Among the characteristic findings using this approach are: (i) morphometric alterations in the mitochondria and melanosomes of melanoma cells treated with azelaic acid; (ii) deregulation of mitochondrial control and extramitochondrial metabolism in similarly treated cells; (iii) considerable acceleration of NAD(P) transient kinetics in atractylate-treated L sarcoma cells; (iv) alterations of mitochondria and Golgi in fusion-deficient myoblasts; (v) tentative recognition of beta-glucosidase deficiency in Gaucher disease cells by the use of fluorescent and fluorogenic lysosomal probes; and (vi) UVA-induced accumulation of Schiff bases (a kind of accelerated photo-aging) in yeast and kidney epithelial cells. Because these studies utilize probing at whatever points along the concerned pathways become accessible, at first glance they may look disconnected. What and where is the connecting thread, for instance, between studying melanoma metabolism, melanosome morphometry, hepatocyte organelle morphogenesis and transformation, myotube organelle morphogenesis and fusion-non-fusion, and lysosomal activity in gene-deficient cells? In the mapping of the regulatory and deregulatory mechanisms involved in the switching of differentiation or transformation, each of the above topics carries an information content towards resolution of the pleiotropic puzzle. The integration of such information with increasing resolution and access to intracellular microdomains may ultimately allow focus on the precise target, the switch from differentiation to transformation or vice versa.

The influence of rutin on the weight, metastasis and melanin content of B16 melanotic melanoma in C57BL/6 mice.

The influence of rutin on the growth rate and tumor weight of B16 melanoma as well as melanin content and ultrastructure of melanoma cells and metastasis formation was studied in mice. All mice were injected s.c. into the left flank with 0.2 ml of suspension containing 10(6) B16 melanoma cells. The experimental groups were treated with a solution of rutin i.p. every two days with total doses of 1, 5 or 10 mg/mouse. The rutin was dissolved in DMSO. The control groups of mice were injected with 0.2 ml 0.9% NaCl and 0.2 ml DMSO. Increasing doses of rutin 1, 5 or 10 mg per mouse caused augmentation of the tumor mass to 2400 mg, 2600 mg and 2800 mg respectively, whereas the tumor weight of the control group was 980 mg. The median number of lung metastases in the control groups was 12; after treatment with 5 or 10 mg of rutin, the number of lung colonies increased to 19 and 27, respectively. The administration of 10 mg rutin inhibited melanin formation by about 43%. The melanosomes in the experimental groups were in the 2nd or 3rd stage, and the low content of melanin was noticed.

Werner syndrome: studies in an affected family reveal a cellular phenotype of unaffected siblings.

Werner syndrome is an inherited disease with symptoms of presenescence. The primary defect site either on the protein or at the DNA level is not known, nor is it possible to identify a heterozygous phenotype. On the basis of cellular peculiarities expressed in the homozygotes-lifespan reduction of cells in culture, length of population doubling time and chromosomal instability-we searched for a 'Werner-like' phenotype in otherwise phenotypically unaffected siblings. We established primary fibroblasts from eight members of a Tyrolean family, two of whom had been diagnosed as typical Werner syndrome, as well as from unrelated healthy young and old volunteers. Determination of the lifespan of each strain and studies on population doubling time and chromosomal instability revealed similar cellular characteristics in all family members, albeit to a lesser extent with the siblings than with the homozygotes when compared to age-matched controls. These features, also apparent in cultivated fibroblasts from old but healthy controls, appear to be indicative of Werner syndrome when expressed in young or middle aged persons. The possible identification of otherwise clinically healthy gene carriers of Werner syndrome is of utmost importance for genetic counselling and medical surveillance for this disorder.

[The inhibition of the proliferation and energy metabolism of Cloudman melanoma cells in vitro by aphidicolin and gamma radiation]. / Hemmung der Proliferation und des Energiestoffwechsels von Cloudman-Melanomzellen in vitro durch Aphidicolin und Gammastrahlung.

PURPOSE: The present investigation is concerned with the effect of aphidicolin (a tetracyclic diterpenoid) and gamma-ray [correction of X-ray] irradiation--alone or in combination--on cell proliferation, protein production, glucose consumption and lactate production of in vitro cultured Cloudman melanoma cells. METHODS: Monolayer cultures of Cloudman melanoma cells in exponential growth phase were irradiated with 2, 4 or 6 Gy (cobalt-60) or treated for 96 h with 0.1, 0.2 or 1.0 microgram/ml aphidicolin. Further, the combination of 0.1 or 0.2 micrograms/ml aphidicolin with the above mentioned radiation doses were investigated. Measurements were performed in regard to changes of cell number, protein content, glucose consumption and lactate production. RESULTS: Single treatments resulted in a dose-dependent inhibition of cell proliferation, protein production, glucose consumption and lactate production. Combined treatment with aphidicolin and irradiation caused an augmentation of the respective single effects. The inhibitory effects were more pronounced under serum-free than serum-containing culture conditions. Influence of aphidicolin and irradiation did not result in an immediate cell death, but in a state of unbalanced growth with enlarged cells (in part with an increased number of nuclei) and an increased cellular protein content. CONCLUSIONS: The combination of aphidicolin with gamma-ray [correction of X-ray] irradiation caused a stronger effect than the respective single treatments.

[Decreased stimulation of hyaluronic acid synthesis by PDGF, IGF-I or serum in the aging process of skin fibroblasts in vitro]. / Verminderte Stimulierbarkeit der Hyaluronsäuresynthese durch PDGF, IGF-I oder Serum in der Alterungsphase von Hautfibroblasten in vitro.

Human skin fibroblasts of phase II ("young" cells derived from populations with a low population doubling level) and of phase III ("old" cells from populations, which were approx. 2 population doublings before the last possible subculture) were kept under subconfluent conditions in a defined serum-free medium. Thereby the cells are in a non-proliferative "quiescent" state. Glycosaminoglycan (GAG)- and especially hyaluronan (HA)-synthesis and release into the medium were investigated by the incorporation rate of 14C-glucosamine. About 95% of the synthesized (48 h) GAGs and HA were medium-released and 5% cell-bound. HA synthesis rate of phase III-cultures was significantly reduced, as compared with phase II-cultures. Stimulation of HA-synthesis of phase III-cells--in comparison with phase II-cells--by serum, PDGF or IGF-I was strongly reduced. While HA-synthesis of phase II-cells was maximally stimulated by 5% FCS or 20 ng/ml PDGF, phase III-cells dit not exhibit a saturation kinetics up to 20% FCS or 60 ng/ml PDGF. The strongly reduced HA-synthesis rate of phase III-cells--compared with phase II-cells--in the non-stimulated quiescent state as well as after stimulation by PDGF, IGF-I or serum might be considered as a biomarker of in vitro (and in vivo?) ageing.

[Electron microscopic studies of the effect of x-rays and L-3,4-dihydroxyphenylalanine (L-Dopa)--alone and in combination-- on Harding-Passey melanoma cells in monolayer cell culture]. / Elektronenmikroskopische Untersuchungen zur Wirkung von Röntgenstrahlen und L-3,4-Dihydroxyphenylalanin (L-Dopa)--allein und inKombination--auf Harding-Passey-Melanomzellen in Monolayerzellkultur.

Monolayer cultures of Harding-Passey melanoma cells in exponential growth phase were exposed to 8 or 16 Gy by X-ray treatment. The 8 Gy treated cells revealed little ultrastructural changes, while the 16 Gy exposed cells showed increased damage as segregates, swollen mitochondria and vacuoles. Sole treatment with L-3,4-dihydroxyphenylalanine (2 x 10(-4) M L-Dopa) resulted in insignificant electronmicroscopically tangible cell alterations. Combined treatment--starting with 8 Gy irradiation followed by a six-day incubation in the presence of 2 x 10(-4) M L-Dopa--revealed more pronounced cell damage with final cell disintegration; the cytoplasm contained an increased number of vacuoles and segregates, a strongly decreased endoplasmic reticulum as well as swollen mitochondria and less pinocytosis vesicles; the cell surface showed less microvilli. Melanin containing organelles increased after the combination treatment. The growth inhibitory and cell destructive influence of L-Dopa on X-ray pretreated melanogenic melanoma cells was explained with the formation of cytotoxic oxidation products of L-Dopa.

Stimulatory effects of ascorbic acid on hyaluronic acid synthesis of in vitro cultured normal and glaucomatous trabecular meshwork cells of the human eye.

The outflow of aqueous humor of the primate eye occurs across the filter system of the trabecular meshwork (TM) into Schlemm's canal. Cells of TM derived from a normal (TM-N-cells) and a glaucomatous human eye (TM-G-cells) were established in monolayer culture. The present comparative experiments were performed with cells kept in a defined serum-free medium (the aqueous humor is nearly protein-free!). Under these conditions the cells stay alive for several months in a non-proliferating state. TM-G-cells exhibited a lower synthesis rate of glycosaminoglycans-especially of hyaluronic acid (HA)--than TM-N-cells. Addition of 50-200 micrograms/ml ascorbic acid (the aqueous humor is characterized by a high ascorbic acid concentration of about 150 micrograms/ml) to the culture medium resulted in a significant dose-dependent stimulation of HA-synthesis and secretion, which was relatively stronger in case of TM-G-cells than with TM-N-cells. Thus, the results suggest a role of ascorbic acid in the probably membrane-localized HA-synthesis. Functions of ascorbic acid and HA for the morphological and functional integrity of the TM-cells in vitro and the outflow apparatus in vivo were discussed.

[Electron microscopic studies on the effects of irradiation and mitoxantrone hydrochloride (Novantrone), alone and in combination, on cells of Harding-Passey melanoma in monolayer culture]. / Elektronenmikroskopische Untersuchungen zur Wirkung von Röntgenstrahlen und Mitoxantron-Hydrochlorid (Novantron)--allein und in Kombination--auf Harding-Passey-Melanomzellen in Monolayer-Kultur.

2 Gy or 4 Gy irradiation of cultured Harding-Passey melanoma cells revealed electronmicroscopically none or little cell damage. 48 hour exposure of cultures to mitoxantrone concentrations of 1 x 10(-8) M or 3.3 x 10(-8) M also exhibited little cell damage. However, combination of these treatments--in comparison with single treatment--resulted in increased cell damage. The present paper describes the extent of Harding-Passey melanoma cell damage following a radiation exposure (2 or 4 Gy) or a mitoxantrone treatment (1 x 10(-8) M or 3 x 10(-8) M) for 48 hours alone or in combination.