Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

Induction of antitumor immunity by indomethacin

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E₂ (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2. Received: 12 August 1999 / Accepted: 21 September 1999     

αvβ3 expression on blood vessels and melanoma cells in primary lesions; differential association with tumor progression and clinical prognosis

The α_vβ₃ integrin has emerged as a key mediator in angiogenesis. Its role in tumor-induced angiogenesis is supported by its up-regulation in vivo in the vasculature of a number of different types of carcinoma. The potential clinical significance of α_vβ₃ expression on blood vessels in carcinomas is suggested by its association with tumor progression. Currently no information is available about the clinical significance of α_vβ₃ expression on the vasculature of lesions of melanocytic origin. Since we have previously found that α_vβ₃ expression on melanoma cells in primary lesions is associated with a poor prognosis, in the present study we have compared α_vβ₃ expression on blood vessels and on cells of melanocytic origin in nevi and in malignant melanoma lesions. In addition we have examined the lesions for expression of the α_v subunit to gain information on the regulation of α_vβ₃ expression on endothelial cells and on cells of the melanocyte lineage. α_vβ₃ expression on endothelial cells and on melanocytic cells was a relatively sensitive and specific marker for malignant lesions. However, α_vβ₃ expression on endothelial cells in primary melanoma lesions was not associated with the prognosis of the disease. The α_v subunit and the α_vβ₃ complex were differentially expressed on endothelial cells and on melanocytic cells, implying that different regulatory pathways control their expression. This finding may account for the differential clinical significance of α_vβ₃ expression on tumor vasculature and on melanoma cells we observed in our patient cohort. Lastly, α_vβ₃ may be a useful target for immunotherapeutic approaches in melanoma because of its high expression on the vasculature of all metastatic lesions tested and its restricted distribution in normal tissues. Received: 18 February 2000 / Accepted: 12 April 2000     

CD4+ T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ). We have previously demonstrated that peptide-specific CD4⁺ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. Methods: Two HLA class II⁺ melanoma cell lines derived from metastases were co-cultured with a human CD4⁺ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specific CD4⁺ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4⁺ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNγ. Received: 1 July 1999 / Accepted: 17 September 1999     

Stereological Studies of the Effects of α-MSH and cAMP on Melanosomes in Melanoma Cells

The effects of α-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing α-MSH or cAMP harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V_Vpm,cell’V_pm) and mature melanosomes (V_Vmm,cell’V_mm) and the number of mature melanosomes (N_mm). Both stimulators also caused declines in the volume of individual mature melanosomes (_Vimm) the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAME The effect cAMP on some parameters occurred 24 hours prior to α-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enchanced melanogenesis by increasing the V_Vpm,cell’V_Vmm,cell’V_pm, V_mm and N_mm. The melanogenic level did not depend on the _Vimm and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAME It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to α-MSH.     

Thrombospondin‐1 is part of a Slug‐independent motility and metastatic program in cutaneous melanoma,in association with VEGFR‐1 and FGF‐2

Differently from most transformed cells, cutaneous melanoma expresses the pleiotropic factor thrombospondin‐1 (TSP‐1). Herein, we show that TSP‐1 (RNA and protein), undetectable in four cultures of melanocytes and a RGP melanoma, was variously present in 13 cell lines from advanced melanomas or metastases. Moreover, microarray analysis of 55 human lesions showed higher TSP‐1 expression in primary melanomas and metastases than in common and dysplastic nevi. In a functional enrichment analysis, the expression of TSP‐1 correlated with motility‐related genes. Accordingly, TSP‐1 production was associated with melanoma cell motility in vitro and lung colonization potential in vivo. VEGF/VEGFR‐1 and FGF‐2, involved in melanoma progression, regulated TSP‐1 production. These factors were coexpressed with TSP‐1 and correlated negatively with Slug (SNAI2), a cell migration master gene implicated in melanoma metastasis. We conclude that TSP‐1 cooperates with FGF‐2 and VEGF/VEGFR‐1 in determining melanoma invasion and metastasis, as part of a Slug‐independent motility program.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.     

The number distribution for involved lymph nodes in cancer

The number of involved lymph nodes exhibits considerable heterogeneity within populations. Here, the implications of population heterogeneity are explored with respect to the kinematics of nodal metastases. Data from the National Cancer Institute's Surveillance, Epidemiology, and End Results program for 224656 breast, 12404 gastric, 18015 rectal, 4117 cervical and 2443 laryngeal cancers as well as 9118 melanomas were used to construct frequency distributions for the number of involved nodes which were then fitted to the negative binomial distribution. The negative binomial distribution described the heterogeneity in nodal involvement well. The patterns of nodal involvement can be explained by either of two models: one where involved nodes could seed further nodal metastases, the other where the number of nodal metastases in any individual was randomly distributed, with the deviations between patients accounted for by population heterogeneity. Since the number of sampled nodes similarly approximated a negative binomial distribution, random involvement with superimposed population heterogeneity would more credibly explain both sets of observations.     

Interleukin 21 therapy increases the density of tumor infiltrating CD8<Superscript>+ </Superscript>T cells and inhibits the growth of syngeneic tumors

Interleukin (IL)-21 is a recently discovered cytokine in early clinical development, which has shown anti-tumor activity in various animal models. In the present study, we examine the anti-tumor activity of IL-21 protein therapy in two syngeneic tumor models and its effect on the density of tumor infiltrating T cells. We treated mice bearing established subcutaneous B16 melanomas or RenCa renal cell carcinomas with intraperitoneal (i.p.) or subcutaneous (s.c.) IL-21 protein therapy and subsequently scored the densities of tumor infiltrating CD4⁺ and CD8⁺ T cells by immunohistochemistry. Whereas both routes of IL-21 administration significantly inhibited growth of small, established RenCa and B16 tumors, only s.c. therapy significantly inhibited the growth of large, established tumors. We found a greater bioavailability and significant drainage of IL-21 to regional lymph nodes following s.c. administration, which could account for the apparent increase in anti-tumor activity. Specific depletion of CD8⁺ T cells with monoclonal antibodies completely abrogated the anti-tumor activity, whereas NK1.1⁺ cell depletion did not affect tumor growth. In accordance, both routes of IL-21 administration significantly increased the density of tumor infiltrating CD8⁺ T cells in both B16 and RenCa tumors; and in the RenCa model s.c. administration of IL-21 led to a significantly higher density of tumor infiltrating CD8⁺ T cells compared to i.p. administration. The densities of CD4⁺ T cells were unchanged following IL-21 treatments. Taken together, these data demonstrate that IL-21 protein has anti-tumor activity in established syngeneic tumors, and we show that IL-21 therapy markedly increases the density of tumor infiltrating CD8⁺ T cells.