摘要:

Melanomas have been induced in hamsters and guinea pigs with short-term, low dose applications of dimethylbenz [a]anthracene (DMBA) alone. In mice, however, melanoma induction has required either croton oil or ultraviolet radiation promotion in addition to DMBA. In this study, we report the development of a malignant melanoma, with metastases, in a hairless mouse after six applications of 0.25% DMBA alone. At sacrifice, a large primary tumour with characteristics of intralesional transformation was present, along with numerous pigmented macules and papules. Metastases were present in lymph nodes and lungs. There was a marked similarity between this melanoma and its precursor lesions and those seen in an earlier, Weiser-Maple guinea pig model, which, in turn, resembled human melanoma.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-γ (IFNγ)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine ± IFNγ (VAC+, VAC−); (ii) syngeneic 2 × 106G3.12 cells plus DETOX (Syn+D), (iii) 2 × 106allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC− and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P< 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNγ incubation did not increase the effect in either the challenge cells or the vaccine cells (P> 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%;P< 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%;P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNγ under these conditions does not augment protection.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Among the various types of human interferons, human interferon-β (HuIFNβ) has the strongest anti-proliferative activity against human melanoma cell lines. Therefore, we investigated the growth inhibitory effect of a cationic liposome containing the HuIFNβ gene on human melanoma cell linesin vitroandin vivo. After transfection with liposomes containing the HuIFN-β gene, human melanoma cell lines produced HuIFNβ in the culture medium at levels ranging from 67 to 3.8 IU/ml on day 6, and growth of the cells was inhibited by 71–92%. Moreover, six injections of liposomes containing the HuIFNβ gene completely eradicated human melanoma nodules transplanted onto the backs of nude mice 40 days after the first injection. Histological analysis of the injected nodules revealed that the HuIFNβ gene transfection induced apoptosis of the human melanoma cells. These data suggest that transfection of the HuIFNβ gene using cationic liposomes is a promising candidate for gene therapy of human melanoma.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Inactivation of p16INK4aand/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. ActivatingCDK4mutations prevent the binding and inhibition of CDK4 by p16INK4a. A second, more indirect role for CDK4 is in late G1, where it may sequester the inhibitors p27KIP1or p21CIP1away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G1into S phase. As the pivotal residues around the most predominant R24C activatingCDK4mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27KIP1or p21CIP1. However, except for a silent polymorphism, we detected no variants within this region of theCDK2gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of theCDK2gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigenpmel17, which maps less than 1 kb away in head to head orientation withCDK2and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

During the initiation and progression of malignant melanoma, a series of different genetic events accumulate on several different chromosomes. The biological heterogeneity of tumour cells presents a major problem, preventing effective treatment of melanoma. To examine the degree of genetic heterogeneity, we searched for allelic losses (loss of heterozygosity; LOH) on chromosomes 9p, 9q, 1p and 17p, examining different areas within human melanoma metastases. All of the examined metastases were informative within at least one dissected area for at least one marker. Out of 29 areas in 11 melanoma metastases, 58% showed LOH with at least one marker. On chromosome 9p21–22, eight out of 26 informative loci (31%) showed LOH at D9S171 (three not informative), two out of 18 (11%) at IFNA (11 not informative) and seven out of 24 (29%) at D9S169 (five not informative). LOH on chromosome 9q22.3 was examined by the microsatellite marker D9S12; three out of 24 areas (12.5%) showed LOH, and five were not informative. Deletions on chromosome 1p were assessed using D1S450. Four out of 25 (16%) showed LOH; four were not informative. Deletions on chromosome 17p13 were examined with TP53; two out of 21 cases (9%) showed LOH, and eight were not informative. Our data demonstrate an impressive heterogeneity of allelic losses in the investigated chromosomal areas within the same metastatic lesion. This suggests that there is not one specific genetic alteration that accounts for melanoma progression to metastases. Rather there seem to be multiple genetic alterations accumulating even on the same chromosome, and progression from melanoma to metastases is paralleled by the accumulation of clones harbouring multiple genetic abnormalities.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

As with most cancers, the aetiology of human cutaneous melanoma is likely to be multifactorial and to include the accumulation of irreversible alterations in an unknown number of genes. Elucidating this molecular progression necessitates both the identification of genetic perturbations at each clinically relevant stage, and the assessment of their impact on the normal melanocyte. The observation that the epidermal melanocyte, in contrast to metastatic melanoma cells, requires activation of the protein kinase C (PKC) pathway to facilitate growthin vitroindicates that one or more isoforms (or substrates) of this large and complex family of proteins are among those that undergo alteration during the development of malignant melanoma. Consequently, a number of studies have investigated the expression of various PKC family members in both melanocyte and melanoma cell lines, without a consensus of opinion as to which isoforms are of biological significance in melanoma development and progression. The present study involved a comprehensive evaluation of the PKC profile in normal melanocytes and in 16 metastatic melanoma cell lines. The results show that the major difference in isoform expression between epidermal melanocytes and melanoma cells is the loss of PKCβ protein expression in 90% of melanoma cell lines. Examination of PKCβ in benign and malignant melanocytic lesions revealed that this protein is either downregulated or absent in both naevi and metastatic melanomas. We conjecture that, although the loss of PKCβ expression is a common phenomenon in malignant melanocytes, it may be related more to a normal process of melanocytic differentiation than to malignant transformation.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

For the molecular detection of rare tumour cells in clinical samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) offers two important advantages over conventional RT-PCR assays: the results are quantitative and, perhaps more importantly, it facilitates exact sensitivity controls on a per sample basis as well as exact comparison of different assay protocols. We report here on quantitative results obtained with different protocols for RNA isolation and cDNA synthesis for amplification of β2-microglobulin transcripts using the light cycler system. Furthermore, housekeeping gene-specific PCRs were compared with PCRs specific for an artificial transcript (internal standard) detected simultaneously at a level comparable to the wild-type sequence. Artificial tyrosinase transcripts derived from a vector construct stably transfected into a human lymphoma cell line were used as a model to test the usefulness of artificial internal standards as an alternative to housekeeping genes. The highest RNA yields were obtained using a combination of phenol-chloroform extraction and the High Pure RNA Isolation Kit. Analysing β2-microglobulin transcript-specific RT-PCRs, the highest sensitivity was obtained for cDNAs generated with Omniscript reverse transcriptase and oligo-p(dT)15primer. Regarding patient blood samples, RT-PCRs specific for β2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts provided quantitative data for all, for 18 out of 21, and for 10 out of 21 samples, respectively. Quantification of β2-microglobulin transcripts by the light cycler system defined the protocol revealing the highest cDNA quality. Comparisons of quantitative data from RT-PCRs specific for β2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts enabled us to determine a close range for crossing points within which sufficient cDNA quality can be guaranteed, even for the detection of rare transcripts. PCRs specific for the artificial internal standard are ideally suited for cDNA quality assessment on a per sample basis.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

In this study procaspase expression levels were investigated by Western blotting in a panel of established melanoma cell lines, transformed melanocytic cell lines and normal primary melanocytes. Upstream caspases such as procaspase-8 that contain a death effector domain were found to be overexpressed in transformed melanocytes and melanoma cell lines compared with melanocytes. Heterogeneous levels of procaspase-8 were seen in melanoma cells, including one cell line that completely lacked procaspase-8 expression. Procaspase-10 is generally overexpressed in transformed melanocytes and melanoma cell lines. Expression of the downstream procaspases-3 and -7 was increased in melanoma cells compared with normal melanocytes. Procaspases containing caspase recruitment domains such as procaspase-2 were expressed at similar levels in nearly all the cell lines investigated. Reduced levels of procaspase-1 compared with normal melanocytes were detected in transformed melanocytes and melanoma cell lines. These data indicate that procaspase levels in general increase during the malignant transformation of melanocytic cells.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The cytokines interleukin (IL)6 and IL10 appear to be involved in the progression of melanoma because they are secreted by malignant cells and their serum levels are associated with poor survival and with advanced stages of the disease. Antitumour immunity is considered to be a T-cell response, mediated mainly by type 1 cytokines such as IL12 and interferon-γ (IFNγ). We evaluated the serum levels of cytokines involved in the host response against tumour (IL12, IFNγ) and/or the progression of melanoma (IL6, IL10) in 45 melanoma patients with localized and metastatic disease and in 45 controls, using commercially available enzyme-linked immunosorbent assay (ELISA) kits. In the controls, IL6 and IL12 were nearly undetectable, whereas the IL10 and IFNγ ranges were 0.5–9 pg/ml and 2–4.8 pg/ml, respectively. In the melanoma patients, pathologically high values were found in 44.4% for IL6, in 24.4% for IL10, and in 60% for IL12. Significantly higher values were found for IL6 and IL12, and lower values for IFNγ. This study highlights a significant difference in serum cytokine profiles between controls and melanoma patients, which is mainly due to the high levels of IL6 and IL12 and the low levels of IFNγ.

ISSN:0960-8931    

出版商:OVID    

年代:2001

数据来源: OVID