Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt

There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.     

Possible involvement of DNA methylation in 5-azacytidine-induced neuronal cell apoptosis

Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052-1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in microM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in microM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line

A new neuronal cell line was generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus-2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Addition of dopamine to 13.S.1.24 cultures induced reduction of cell number within 2 days. Two hallmarks of apoptosis were detected in dopamine-treated cultures: internucleosomal DNA fragmentation and nuclear condensation. Dopamine did not alter the cell proliferation rate, as assessed by [3H]thymidine incorporation. Dopamine also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein-immunoreactive neurons. Time-dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by dopamine were concomitant. Both apoptosis and differentiation triggered by dopamine were dose-dependent, half-maximal effects being obtained with approximately 10 microM dopamine. Mediation of both effects by dopaminergic D2 receptors was supported by several observations: active dopamine doses in micromolar ranges, quinpirole agonism and eticlopride antagonism, D2-characteristic rank order of potency among the three agonists tested, and specific binding of a selective D2-like radioligand to 13.S.1.24 cells. The present data altogether indicated that dopamine commits immortalized olfactory neuronal cells in vitro either to apoptosis or to olfactory-like differentiation via D2 dopaminergic receptors.     

Induction of apoptosis by organotin compounds in vitro: neuronal protection with antisense oligonucleotides directed against stannin

Immortalized cell lines and primary neuronal cultures were used to characterize the selective toxicity of trimethyltin (TMT),triethyltin (TET) and tributyltin (TBT). TBT and TET were cytotoxic at similar concentrations in the immortalized cell lines tested; the 50% toxic concentration (TC50) was 1 to 11 microM. In contrast, immortalized cell lines varied considerably in their sensitivity to TMT, with sensitive cell lines (neuroblastomas, T-, B-cell lines) showing TC50 values of 2 to 8 microM, whereas insensitive cells (NIH-3T3 fibroblast, HTB-14 glioma, TC-7 kidney cells) had TC 50 values > 100 microM. Primary neuronal cell cultures were very sensitive to organotins (TC50 values, 1-10nM), and showed patterns of selective toxicity with respect to neuronal and glial cells. Because organotin toxicity evolves over 24 to 48 hr. we determined whether these compounds induced apoptosis in primary cultures. TMT increased (P < .05) the fraction of apoptotic cells 6 and 12 hr after treatment with TMT at TC50 concentrations. Prior studies suggested that a protein, stannin, was localized in cells sensitive to organotins. Stannin was expressed in several TMT-sensitive cell lines (PC12, T, B cells) and in primary neurons in culture. Stannin was absent in the resistant HTB-14 glioma cell line. The role of stannin in mediating TMT toxicity in primary cultures was investigated by blocking stannin expression with specific antisense oligonucleotides. Treatment of primary cultures with antisense oligonucleotides for 48 hr before and during TMT treatment significantly protected neurons from the neurotoxic and apoptotic effects of TMT. This effect was not observed with scrambled oligonucleotide controls. Thus, TMT may induce apoptosis in sensitive cells, which is partly mediated by stannin. Based on the available data we conclude that stannin expression is necessary, but not sufficient for TMT toxicity.     

Induction of apoptosis in human cancer cell lines by the novel anthracenyl-amino acid topoisomerase I inhibitor NU/ICRF 505

A 77-year-old woman with a productive cough and fever was admitted to the hospital. Pulmonary and endobronchial tuberculosis, pneumonia of the left upper lobe, and stenosis of the left main bronchus were diagnosed. She was given the antimycobacterial drugs isoniazid, rifampin, and streptomycin, and her condition improved. Two months later, bronchoscopy revealed semilunar-shaped stenosis of the left main bronchus, and auscultation revealed wheezing in the middle-end expiratory phase. A continuous flow murmur (Levine III) was also heard at the left anterior chest wall. Cardiac catheterization with subclavian arteriography revealed two left subclavian-pulmonary shunts. In a case of systemic-pulmonary shunt such as this, the bronchial stenosis could be surgically repaired, but the result would be an increase in dead space. If left untreated, the pulmonary hypertension would progress and symptoms of pulmonary disease would become more severe. Subclavian-pulmonary artery shunt is a very rare complication of pulmonary tuberculosis. Surgical treatment should consist of open bronchoplasty along with lobectomy and removal of the shunt, rather than embolization of the shunt and endoscopic bronchoplasty.     

Induction of apoptosis by 2-chlorodeoxyadenosine in B cell chronic lymphocytic leukemia

We investigated whether 2-chlorodexoyadenosine could induce apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells in vitro using clinically achievable drug doses, measuring apoptosis ratio by flow cytometry. B cells were isolated from previously untreated patients and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with 2-chlorodeoxyadenosine at different concentrations, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of drug, but the level of apoptosis was greater in cells treated with 2-chlorodeoxyadenosine after the second day of culture. The present in vitro study of B-CLL cells from previously untreated patients suggests this chemotherapeutic agent activates a program of cell death by apoptosis using a drug dose equivalent to the physiological concentration used in patients in vivo. These data reveal an interesting possibility in the 2-chlorodeoxyadenosine treatment of untreated patients by neoplastic B cell apoptosis induction.     

Role of nitric oxide in methamphetamine neurotoxicity: protection by 7-nitroindazole, an inhibitor of neuronal nitric oxide synthase

The role of nitric oxide (NO.) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 x 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO. formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO. may be directly involved in dopaminergic terminal damage.     

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.     

Induction of apoptosis in leukemia U937 cells by 5'-deoxy-5'-methylthioadenosine, a potent inhibitor of protein carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5'-deoxy-5'-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer

Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 = 2.8-3.7 microM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 = 3.5-11.2 microM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 microM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents.     

Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis

Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell "suicide" whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6-hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricarboxylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations.     

Geminin, an inhibitor of DNA replication, is degraded during mitosis

We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). We propose that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.     

Induction of Fas-mediated apoptosis in a human renal epithelial cell line by interferon-gamma: involvement of Fas-mediated apoptosis in acute renal rejection

To examine the role of the Fas ligand/Fas (FasL/Fas) system and apoptosis in renal allograft rejection, we analyzed FasL/Fas expression and apoptosis in 63 renal allograft biopsy specimens obtained from 56 renal transplant recipients in Tokyo, Japan. Sixteen biopsy specimens were diagnosed (Banff criteria) as acute rejection (AR), 17 as AR plus chronic rejection, 10 as borderline stages, and 20 as no rejection (NR). Immunohistochemically, Fas antigen was highly expressed in the renal tubular epithelium in tissues from patients with rejection. The mean number of Fas-positive tubular epithelium was significantly higher in biopsy specimens with AR than in those with NR, but FasL expression was highly expressed in infiltrating lymphocytes in the interstitium of allografts with cellular rejection. The mean number of FasL-positive infiltrating lymphocytes was significantly higher in specimens with AR than in those with NR or borderline stages. For detection of apoptotic cells, the specimens were subjected to terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, which showed that the mean number of tubular epithelial cells positive for this labeling was highest for the specimens with AR, the number being significantly higher than in those with AR plus chronic rejection or with NR. Thus, Fas expression on epithelial cells might actively trigger their apoptosis by the interaction between Fas and FasL. Studies of human normal renal-derived cell lines (RPTEC 2601 [epithelial] and NHMC 5155 [mesangial]) showed that both constitutively expressed Fas (but not FasL) mRNA. After pretreatment with interferon-gamma, Fas-induced apoptosis was observed in the RPTEC 2601 line, but without interferon-gamma pretreatment, anti-Fas-mediated apoptosis was not seen. Under identical conditions, the NHMC 5155 line was resistant to anti-Fas-mediated apoptosis regardless of interferon-gamma pretreatment. This suggested that AR might be associated with increased apoptosis in the renal tubular epithelial cells mediated by the Fas/FasL system.     

Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine

The prototype of a new class of antiproliferative phospholipid analogs, hexadecylphosphocholine (HePC), has been shown to inhibit tumor growth and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC, e.g. protein kinase C and CTP:phosphocholine cytidylyltransferase, have been proposed, the mechanisms of HePC-induced anticancer activity are still unclear. Considering that the antiproliferative effect of HePC correlates with inhibition of phosphatidylcholine biosynthesis, which is tightly coupled to sphingomyelin biosynthesis, we tested the hypothesis that treatment of cells with the anticancer drug leads to increased cellular ceramide and subsequently to apoptotic cell death. In the present study, we showed that 25 micromol/liter HePC induced apoptosis. In further experiments, we demonstrated that HePC inhibited the incorporation of radiolabeled choline into phosphatidylcholine and at a later time point into sphingomyelin. This was confirmed by metabolic labeling of the lipid backbone using radiolabeled serine, and it was shown that HePC decreased the incorporation of serine into sphingomyelin by 35% and simultaneously increased the incorporation of serine into ceramide by 70%. Determination of the amount of ceramide revealed an increase of 53% in HePC-treated cells compared with controls. In accordance with the hypothesis that elevated ceramide levels may be the missing link between the metabolic effects of HePC and its proapoptotic properties, HePC-induced apoptosis was blocked by fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, we found that membrane-permeable ceramides additively increased the apoptotic effect of HePC.     

Induction of apoptosis in cardiac myocytes by an A3 adenosine receptor agonist

The effects of the selective adenosine (ADO) A3 receptor agonist IB-MECA (N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide) on cultured newborn rat cardiomyocytes were examined in comparison with ADO, the ADO A1 receptor-selective agonist R-PIA (N6-R-phenylisopropyladenosine), or the ADO A3 selective antagonist MRS 1191 (3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5 dicarboxylate), using digital image analysis of Feulgen-stained nuclei. At high concentration, IB-MECA (>/=10 microM ) and ADO (200 microM) induced apoptosis; however, R-PIA or MRS 1191 did not have any detectable effects on cardiac cells. In addition, DNA breaks in cardiomyocytes undergoing apoptosis following treatment by IB-MECA were identified in situ using the nick end labeling of DNA ("TUNEL"-like) assay. In the presence of >/=10 microM IB-MECA, disorder in the contraction waves appeared, and a decrease in the frequency of beats was observed. Analysis with light microscopy revealed that the number of contracting cells decreased in a concentration-dependent manner. The A3 receptor agonist IB-MECA caused an increase in intracellular free calcium concentration ([Ca2+]i). The drug produced a rapid rise followed by a sustained increase in [Ca2+]i, which lasted for 40-60 s. Finally, cessation of beating and Ca2+ transients were observed. Full recovery of contractile activity and rhythmical Ca2+ transients were observed 15-20 min after IB-MECA treatment. The induction of apoptosis in the cardiocytes by IB-MECA led to the appearance of features of apoptotic nuclei: the onset of condensation, compacting, and margination of nuclear chromatin. These effects were accompanied by the disintegration of the structural framework of the nucleus and nuclear breakdown. The results suggest that activation of the A3 adenosine receptor may participate in the process of apoptosis in cardiomyocytes.     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.