DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and α₄β₁ integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and α₄β₁ integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and α₄β₁-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells. J. Cell. Physiol. 177:299–312, 1998. © 1998 Wiley-Liss, Inc.     

Effect of <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> on TIMP-1 secretion from mouse melanoma cell

Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.     

Influence of nitric oxide synthase activity on amino acid concentration in the quinolinate lesioned rat striatum: A microdialysis and histochemical study

Summary. The influence of nitric oxide synthase (NOS) activity on the KCl-evoked amino acid concentrations was investigated by in vivo microdialysis in the striatum in a rat model of excitotoxic lesion. Basal microdialysate levels of amino acids decreased during the quinolinic acid-induced neurodegeneration process, except for glutamine that increased initially and returned to control values 30 days after quinolinic acid exposure. KCl-evoked increase of extracellular amino acid concentration was reduced due to NOS activity in the striatum of both controls and lesioned animals, except for 120 days after quinolinic acid injection. These changes of amino acid concentrations in microdialysates correlated with the known biochemistry of the consecutive domineered cell types during the lesion process as revealed by histochemistry for NOS, NADPH-diaphorase, GFAP and isolectin B4. The present data provide direct evidence that NOS activity can modulate extracellular amino acid concentrations in the striatum not only under physiological conditions, but also during a pharmacologically induced lesion process and, thus, suggests that nitric oxide affects neurodegeneration via this pathway. Received October 20, 1999; Accepted February 25, 2000     

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Teratogenicity of 3-hydroxynorvaline in chicken and mouse embryos

Summary. 3-Hydroxynorvaline (HNV; 2-amino-3-hydroxypentanoic acid), a microbial L-threonine analogue, is toxic to mammalian cells and displays antiviral properties. In view of this, we investigated the toxicity and/or potential teratogenicity of HNV in developing chicken and mouse embryos. HNV was administered to chicken embryos (in ovo; dose 75–300 μmole/egg; 48 h post-incubation) and pregnant Hanover NMRI mice (per os; total dose 900–1800 mg/kg body mass; gestation days 7–9). Control animals received sterile saline solutions. Harvested embryos (chicken embryos, 10 days post-incubation; mouse embryos; gestation day 18) were fixed in glutaraldehyde and stereomicroscopically inspected for signs of dysmorphogenesis. Body mass, body and toe length and mortality of chicken embryos, and the body mass and mortality of mouse embryos were recorded. HNV exposure significantly increased the incidence of embryotoxic (growth retardation, toxic mortality) and congenital defects in both chicken and mouse embryos. All the observed effects were dose-dependent. In conclusion, HNV is an embryotoxic and teratogenic compound, which caused significant developmental delay and congenital defects in developing chicken and mouse embryos.     

The relationship between albumin, other plasma proteins and variables, and age in the acute phase response after liver resection in man

Summary. A large series of plasma albumin (ALB, g/dl) and simultaneous blood and clinical measurements were prospectively performed on 92 liver resection patients, and processed to assess the correlations between ALB, other plasma proteins, additional variables and clinical events. The measurements were performed preoperatively and at postoperative day 1, 3 and 7 in all patients, and subsequently only in those who developed complications or died. In patients who recovered normally ALB was 4.3 ± 0.4 g/dl (mean ± SD) preoperatively, 3.7 ± 0.7 at day 1 and 3, and 3.9 ± 0.4 at day 7. In patients with complications its decrease was more prolonged. In non-survivors it was 3.4 ± 0.4 preoperatively, 3.0 ± 0.4 at day 1, and then decreased further. Regression analysis showed direct correlations between ALB and pseudo-cholinesterase (CHE, U/l, nv 5300-13000), cholesterol (CHOL, mg/dl), iron binding capacity (IBC, mg/dl), prothrombin activity (PA, % of standard reference) and fibrinogen, an inverse correlation with blood urea nitrogen (BUN, mg/dl) for any given creatinine level (CREAT, mg/dl), and weaker direct correlations with hematocrit, other variables and dose of exogenous albumin. An inverse relationship found between ALB and age (AGE, years) became postoperatively (POSTOP) also a function of outcome, showing larger age-related decreases in ALB associated with complications (COMPL: sepsis, liver insufficiency) or death (DEATH). Main overall correlations: CHE = 287.4(2.014)^ALB, r = 0.73; CHOL = 16.5(1.610)^ALB (1.001)^ALKPH, r = 0.71; IBC = 68.6(1.391)^ALB, r = 0.64; PA = 13.8 + 16.0(ALB), r = 0.51; BUN = 21.3 + 20.2(CREAT) – 6.2(ALB), r = 0.91; ALB = 5.0–0.013(AGE) – {0.5 + 0.003(AGE) _COMPL + 0.012(AGE) _DEATH } _POSTOP , r = 0.74 [p < 0.001 for each regression and each coefficient; ALKPH = alkaline phosphatase, U/l, nv 98-279, independent determinant of CHOL; discontinuous variables in italics label the change in regression slope or intercept associated with the corresponding condition]. These results suggest that altered albumin synthesis (or altered synthesis unable to compensate for albumin loss, catabolism or redistribution) is an important determinant of hypoalbuminemia after hepatectomy. The correlations with age and postoperative outcome support the concept that hypoalbuminemia is a marker of pathophysiologic frailty associated with increasing age, and amplified by the challenges of postoperative illness.     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Beneficial activity of Enterococcus faecalis CECT7121 in the anti‐lymphoma protective response

Aims: To study the anti‐tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T‐cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated. Methods and Results: In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose‐dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre‐treated i.p. with Ent. faecalis CECT7121 (2·5 × 10⁸ CFU) were challenged i.p. with LBC cells (1·0 × 10⁶ cells) the day after. On day 30 post‐inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre‐treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re‐challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL‐12 and IL‐10 levels were observed in surviving animals. Conclusions: Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down‐regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re‐challenge. Significance and Impact of the Study: This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of ¹²⁵I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.     

Biochemical Analysis of Metastasis-Related Ax Actin in B16 Mouse Melanoma Cells After Chemical Reversional Modulation and of Tumor Progression-Related A′ Actin in the Ontogeny of Human Malignant Melanoma

To examine the correlation between tumor metastasis and A^x actin in mouse melanoma and between tumor progression and A′, actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of A^x actin. Will an induced decrease in metasasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of A^x actin? We also examined the appearance of A′ actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented A^x actin in vitro. These results suggest that the appearance of A^x actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, A^x actin appearance remained unchanged. Appearance of A′ actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A′ actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Structure-activity investigations of polyamine-anthracene conjugates and their uptake via the polyamine transporter

Summary. A series of polyamine conjugates were synthesized and evaluated for their ability to target the polyamine transporter (PAT) in two Chinese hamster ovary (CHO) cell lines (PAT-active CHO and PAT-inactive CHOMG). This systematic study identified salient features of the polyamine architecture required to target and enter cells via the PAT. Indeed, the separation of charges, the degree of N-alkylation, and the spacer unit connecting the N¹-terminus to the appended cytotoxic component (anthracene) were found to be key contributors to optimal delivery via the PAT. Using the CHO screen, the homospermidine motif (e.g., 4,4-triamine) was identified as a polyamine vector, which could enable the selective import of large N¹-substituents (i.e., naphthylmethyl, anthracenylmethyl and pyrenylmethyl), which were cytotoxic to cells. The cell selectivity of this approach was demonstrated in B-16 murine melanoma cells and normal melanocytes (Mel-A). Three polyamine areas (recognition and transport, vesicle sequestration and polyamine-target interactions) were identified for future research.     

Genomewide RNAi screen identifies protein kinase Cβ and new members of mitogen‐activated protein kinase pathway as regulators of melanoma cell growth and metastasis

A large‐scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole‐genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen‐activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C β (PKCβ) as candidate genes. Knockdown of PKCβ most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCβ showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCβ‐specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCβ‐shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCβ seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16–010 cells grew in circular in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melainin pigment (B16-010 < B16-05 < B16-01 ? B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.     

Polyamine analogues – an update

Summary. The polyamines are growth factors in both normal and cancer cells. As the intracellular polyamine content correlates positively with the growth potential of that cell, the idea that depletion of polyamine content will result in inhibition of cell growth and, particularly tumour cell growth, has been developed over the last 15 years. The polyamine pathway is therefore a target for development of rationally designed, antiproliferative agents. Following the lessons from the single enzyme inhibitors (α-difluoromethylornithine DFMO), three generations of polyamine analogues have been synthesised and tested in vitro and in vivo. The analogues are multi-site inhibitors affecting multiple reactions in the pathway and thus prevent the up-regulation of compensatory reactions that have been the downfall of DFMO in anticancer chemotherapy. Although the initial concept was that the analogues may provide novel anticancer drugs, it now seems likely that the analogues will have wider applications in diseases involving hyperplasia.