Effect of 4-hydroxynonenal on antioxidant capacity and apoptosis induction in Jurkat T cells

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and may have either physiological or pathological significance regulating cell proliferation. We studied some biochemical effects of HNE, at various concentrations (0.1-100 μM), on Jurkat T cells incubated thereafter for 24, 48 and 72 h. HNE at low concentrations significantly enhanced the proliferation index, whereas at higher concentrations progressively blocked cell proliferation. Caspase 3 activity increased significantly at HNE concentrations between 1 and 10 μM and decreased at higher concentrations. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) increased progressively with HNE concentrations, particularly GSH-Px. Glucose-6-phosphate dehydrogenase (G6PDH) showed a different pattern, increasing at low HNE (1-5 μM) concentrations and rapidly declined thereafter. These results show that HNE may induce growth inhibition of Jurkat T cells and regulate the activity of typical antioxidant enzymes. Furthermore, the protective effect of doubling the foetal calf serum still points out the risk that cultured cells undergo oxidative stress during incubation.     

Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c-myc messenger RNA

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK-MEL-109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK-MEL-109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose-dependent way. Treatment with 10 ng/ml of TNF resulted in a two-fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c-myc mRNA was increased five-fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c-myc mRNA.     

Enhancement of the growth of human osteosarcoma cells by human interferon-gamma.

It is well known that interferons inhibit cell growth. However, we found that human interferon-gamma (HuIFN-gamma) enhanced the growth of human osteosarcoma cells, HOS-Y1 cells, in a dose-dependent manner. This enhancing effect was found only under the following conditions: when the cells were precultured for 2 or 3 days and then treated with HuIFN-gamma for 2, 3, or 4 days, and when the cells were seeded at a density of 1,000 or 2,000 cells/well. The degree of enhancement of cell growth was maximum when the cells were precultured at a density of 1,000 cells/well for 3 days and then treated with HuIFN-gamma for 2 days. The enhancing effect of HuIFN-gamma disappeared in the presence of anti-HuIFN-gamma antibody. In addition, it was found that the conditioned medium from HOS-Y1 cells enhanced the growth of HOS-Y1 cells, and that the conditioned medium from HOS-Y1 cells cultured with HuIFN-gamma enhanced the cell growth more than that from cells cultured without HuIFN-gamma. Epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1(TGF-beta 1) did not enhance the growth of HOS-Y1 cells. These results suggest that HuIFN-gamma enhanced the cell growth by augmenting the production of unknown growth factor(s) in HOS-Y1 cells via an autocrine mechanism.     

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.     

Clear cell carcinoma of the human ovary synthesizes and secretes a transferrin with microheterogeneity of lectin affinity

Human ovarian clear cell carcinoma cell line (transferrin (Tf)-non-producer), HAC 2, cells were adapted to grow in chemically defined synthetic medium when the cells were cultured with medium containing 10 μg/ml of insulin at least for 6 months. They synthesized and secreted constantly the 80 kDa protein immunologically similar to human serum Tf(15 ± 12 ng/ml/10⁷ cells/3 days). By sensitive lectin-affinity electrophoresis followed by antibody-affinity blotting technique, a concanavalin A weakly bound or unbound, lentil lectin, a strongly reactive abnormal band, which was rarely found in human serum Tf, was detectable in the Tf synthesized by HAC 2 cells (HACTf). These findings suggest that the HACTf may act as one of the autocrine growth factors and that this heterogeneity of HACTf for lectin affinity is ascribed to differences in the carbohydrate moiety of the Tf.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Anchorage-independent growth of murine melanoma in serum-less media is dependent on insulin or melanocyte-stimulating hormone

α-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.     

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 μM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.     

Disturbance of cell proliferation by two model compounds of lipid peroxidation contradicts causative role in proliferative senescence

Cumene hydroperoxide (Chp), a lipophilic peroxide, and hydroxy-nonenal (HNE), a breakdown product of lipid peroxides, were used as model compounds to assess the effects of lipid peroxidation upon cell proliferation. Amniotic fluid fibroblastlike (AFFL) cells and human diploid skin-derived (HDFL) cells were cultured with the two model compounds and cell proliferation was assayed via bromodeoxyuridine-Hoechst flow cytometry. At low doses Chp elicited an accumulation of cells in the S and G2 phase, while at higher doses the fraction of nonproliferating cells increased as well. Low doses of HNE caused an accumulation of cells in the G1 and G2 phase, whereas an additional increase of cells in S phase and in the nonproliferating fraction was found at an elevated concentration. A delay of onset of proliferation was obtained with both Chp and HNE. Permanent arrests in the S, G2, and G1 compartment are provoked by Chp only when Chp was applied together with serum. HNE, to the contrary, elicited a permanent arrest in the G2 and the G1 compartment even if added to quiescent cell cultures. Additionally, HNE caused a combination of a prolongation of the G1 phase of the cell cycle and an arrest in this compartment, which is reminiscent of cell differentiation. HDFL cells were much more sensitive toward Chp than were AFFL cells, but both cell types showed similar sensitivities toward HNE. We conclude that lipophilic peroxides exert toxic effects upon cell proliferation distinct from the pattern elicited by aldehydic breakdown products of lipid peroxides. The pattern of cell cycle arrest induced by Chp and HNE makes it unlikely that Chp and HNE, or related products of lipid peroxidation, are responsible for the limitation of the proliferative life span of human fibroblasts in culture.     

Protection by thiols of the mitochondrial complexes from 4-hydroxy-2-nonenal

In the present study, the effects of 4-hydroxy-2-nonenal (HNE) on highly purified pyruvate dehydrogenase complex (PDC) and its catalytic components in vitro and on PDC, alpha-ketoglutarate dehydrogenase complex (KGDC), and the branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells were investigated. Among the PDC components, the activity of the dihydrolipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) only was decreased by HNE. Dihydrolipoamide dehydrogenase (E3) protected the E2-E3BP subcomplex from HNE inactivation in the absence of the substrates. In the presence of E3 and NADH, when lipoyl groups were reduced, higher inactivation of the E2-E3BP subcomplex by HNE was observed. Purified PDC was protected from HNE-induced inactivation by several thiol compounds including lipoic acid plus [LA-plus; 2-(N,N-dimethylamine)ethylamidolipoate(.)HCl]. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities, whereas BCKDC activity decreased to a lesser extent. Lipoyl compounds afforded protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. These findings show that thiols, including lipoic acid, provide protection against HNE-induced inactivation of lipoyl-containing complexes in the mitochondria.     

Resistance to tumor growth mediated by Listeria monocytogenes. Destruction of experimental malignant melanoma by LM-activated peritoneal and lymphoid cells.

A murine experimental model of nonspecific tumor destruction mediated by cells activated by Listeria monocytogenes (LM) is described. B16 melanoma growth is prevented or suppressed in the syngeneic host when tumor cells are inoculated in contact with viable LM. In vitro, cultured B16 cells are destroyed by LM immune peritoneal or splenic cells in the presence of the bacterial antigen(s). Activation of LM immune cells in vitro is immunologically specific. Replacement of LM by sheep red blood cells or bovine serum albumin in the in vitro cultures aborts the cytotoxic effect. Further, no tumor cell killing is obtained when thioglycollate-induced or normal peritoneal cells are substituted for LM immune cells in the in vitro cultures. Normal spleen cells in the presence of LM are weakly cytotoxic for B16 cells. Normal peritoneal cells plus LM or LM alone are not. Elimination of thymus derived "T" cells by anti-theta C3H or rabbit anti-mouse brain serum (RAMB) abrogated the cytotoxic effect. Therefore, LM-induced tumor destruction probably occurs through nonspecific mechanism(s) consequent to activation of host "T" cells by specific immune reactivity to LM antigen(s).     

Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.     

Dexamethasone and zinc in combination inhibit the anchorage-independent growth of S-91 Cloudman murine melanoma

Zinc inhibited the colony formation of Cloudman S-91 murine melanoma cells in a dose dependent manner with an ID50 of 3.4 ug/ml. Total inhibition of the melanoma colony-forming units occurred at a zinc concentration of 4.42 ug/ml. In the presence of dexamethasone the ID50 for zinc inhibition was reduced by 49% and total inhibition of anchorage-independent growth occurred at the achievable in vivo zinc concentration of 3.0 ug/ml. Dexamethasone and zinc in combination effected a greater than additive inhibition of the murine melanoma colony-forming units. Statistical evaluation of these results showed that zinc and dexamethasone interacted synergistically to inhibit the formation of murine melanoma colonies.     

Effects of 4-Hydroxynonenal, A Product of Lipid Peroxidation, on Cell Proliferation and Ornithine Decarboxylase Activity

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10^-5 to 10^-6 M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. ³H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.     

Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation

Primary cultures of the medroxyprogesterone acetate-induced mouse mammary tumor line C4-HD are stimulated by medroxyprogesterone acetate (MPA) or progesterone. Serum obtained from ovariectomized, MPA-treated animals (OVX-MPA) exerts a stimulatory effect that is significantly higher than that induced by serum obtained from OVX mice with the exogenous addition of MPA, suggesting the involvement of MPA-induced serum factors potentiating the proliferative effect of MPA. The object of this paper is to further explore the stimulatory effect of mouse serum and to investigate the role of aFGF and bFGF on cell proliferation. The role of PR as possible mediators was tested using two different antiprogestins and antisense oligodeoxynucleotides of PR A isoform. Serum was obtained from OVX untreated or MPA-treated mice and was charcoalized and/or heat-inactivated. The effect of MPA or mifepristone at 10 nM concentrations was tested. Charcoalization and heat inactivation exerted a stimulatory effect (P<0.01) when OVX-serum was used. This effect was potentiated by MPA. Charcoalized OVX-MPA serum induced a significant inhibition of cell proliferation that was restored by the exogenous addition of MPA or by heat inactivation. Mifepristone induced an inhibition of ³H-thymidine uptake when OVX-MPA serum was used. These results suggest that serum factors activated by different manipulations may replace the stimulatory effect of MPA. When charcoalized fetal calf serum (chFCS) was used, a higher proliferative activity was obtained using higher serum concentrations. Mifepristone and onapristone 10 nM also inhibited this effect. aFGF and bFGF 100 ng/ml were both able to stimulate ³H-thymidine uptake. MPA exerted an additive effect. Mifepristone 10 nM inhibited bFGF and MPA+bFGF induced cell proliferation. Antisense oligodeoxynucleotides of PR (ASPR) were used to further confirm the participation of PR in the proliferative pathway of these cells. They inhibited serum and bFGF-induced cell proliferation in a specific dose-dependent manner. Our results suggest that PR play a central role in proliferation and suggest the existence of a cross-talk between steroid and growth factor signaling pathways.     

Possible involvement of 4-hydroxynonenal in splenocyte regulated liver regeneration

Liver regeneration is a complex, systemic process regulated by humoral and cellular mechanisms. Inflammatory response to the extensive tissue damage, as in partial hepatectomy, plays important role during regeneration. Hence, it is assumed that the spleen might play a role in systemic inflammatory response involved in liver regeneration. On the other hand, liver damage and consequential regeneration are often associated with oxidative stress and lipid peroxidation. One of the end products of lipid peroxidation, 4-hydroxynonenal (HNE), is nowadays considered not only as a "second toxic messenger of free radicals" but also as a growth-regulating factor. We therefore studied in vitro interactions of the HNE-treated murine liver cells and autologous spleen cells. The spleen cells supported recovery of liver cells from the HNE cytotoxicity although spleen cells themselves exerted cytotoxic effects against the proliferating liver cells that were not treated with HNE. Our results imply that the cytokines secreted by activated immunocompetent cells may be responsible for the observed recovery of the HNE-damage liver cells, suggesting that HNE might be an important factor regulating cellular and cytokine mediated mechanisms of liver regeneration control.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin