Melanotic Line Research Articles

Role of Interleukins and Nitric Oxide Secretion by Peritoneal Macrophages in Differential Tumoricidal Effect to Transplantable Melanomas as Regarding their Biological Properties

The secretion of interleukins (IL-18, IL-12) and Nitric Oxide (NO) by peritoneal macrophages from hamsters bearing two lines of transplantable melanoma was estimated. Macrophages of animals with melanoma lines secreted less IL-18 but more IL-12 and NO in comparison with the control macrophages. The distinctly higher cytotoxic activity of macrophages from animals with amelanotic line in comparison with the melanotic line was not accompanied by significant differences in the IL-18 and IL-12 secretion between studied groups of macrophages. Thus, it seems that IL-18 play the role in innate immunity but not in adaptive cellular immunity, whereas IL-12 and NO take part in macrophages tumoricidal activity.

Molecular genetic analysis of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood.

Supratentorial primitive neuroectodermal tumours (sPNETs) are malignant central nervous system tumours of childhood which are histologically characterized by poorly differentiated neuroepithelial cells with the capacity for divergent differentiation into glial, neuronal, myogenic or melanotic lines. The histological differential diagnosis between sPNET and glioblastoma multiforme (GBM) may be difficult, particularly as GBMs can sometimes demonstrate a poorly differentiated PNET-like phenotype. To identify molecular genetic markers that may distinguish sPNET and GBM, we investigated 12 cerebral sPNETs and six GBMs from paediatric patients for genetic alterations of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 genes, as well as for allelic loss on chromosome arms 10q and 17p. Mutations of the TP53 tumour suppressor gene were found in one of 12 sPNETs (8%) and two of six GBMs (33%). None of the sPNETs but two of six GBMs (33%, including one GBM with a TP53 mutation) showed allelic losses on chromosome arm 17p. PTEN mutations were detected in one of 12 sPNET (8%) and one of six GBMs (17%). None of the sPNETs and GBMs carried a homozygous deletion involving the CDKN2A tumour suppressor gene. No amplification of the EGFR, CDK4 or MDM2 proto-oncogenes was detected. Taken together, our results indicate that paediatric GBMs differ from sPNETs by a higher incidence of allelic losses on 17p and TP53 mutations. In addition, the patterns of genetic alterations in sPNETs and paediatric GBMs appear to be distinct from those in cerebellar medulloblastomas and adult GBMs, respectively.

Sensitivity of Transplantable Melanoma Cells to Cytokines with Regard to Their Spontaneous Apoptosis

Objectives: Changes in the sensitivity of two lines of transplantable melanoma cells to the antiproliferative activity of interleukin 6 (IL-6), oncostatin M (OSM), tumor necrosis factor-α (TNF-α) during melanoma progression were the subject of this study. We were looking for a correlation between these changes and the ability of melanoma cells to undergo spontaneous apoptosis. Methods: The influence of exogenous cytokines on the proliferation of melanoma cells was measured by colorimetric methods with MTT and apoptosis of these cells was estimated by staining with annexin V/propidium iodide, measurement of DNA degradation and cell cycle analysis. Results: It was observed that during melanoma progression the sensitivity of those two melanoma line cells to the antiproliferative effect of IL-6 and OSM did not change, while a spontaneous alteration of the melanotic line into an amelanotic one seemed to be accompanied by resistance of the amelanotic melanoma cells to the antiproliferative activity of TNF-α. Simultaneously, we observed a decreased ability of amelanotic melanoma cells (in comparison with the native line) to undergo spontaneous apoptosis. Conclusions: The observed resistance to the TNF-α antiproliferative effect which appears during melanoma progression seems to be correlated with a lower ability of the amelanotic melanoma cells to undergo spontaneous apoptosis.

Heterogeneous susceptibility to spontaneous and induced apoptosis characterizes two related transplantable melanomas with different biological properties.

A family of phenotypically and biologically different transplantable hamster melanomas was derived from a single tumor more than 40 yr ago. In this work, we were seeking the differences between the abilities of the cells from two biologically heterogeneous (melanotic and amelanotic) members of this family to undergo spontaneous or camptothecin-induced apoptosis. We studied these differences by looking at three important features of the apoptotic process, i.e. binding of annexin V, DNA fragmentation and caspase-3 activity. Of these, annexin binding and DNA fragmentation were more pronounced in the parental, melanotic line while the activity of caspase-3 was stronger in the amelanotic tumor cells. We concluded that a spontaneous alteration of the original, melanotic melanoma line into an amelanotic one, associated with more aggressive tumor progression, was accompanied by significant decrease in ability to undergo spontaneous and camptothecin-induced apoptosis, and that apoptosis of these two cell types may not depend on the activity of caspase-3.

Angiomorphology of the pigmented bomirski melanoma growing in hamster eye

An melanotic line of Bomirski Hamster Melanoma (BHM Ma) was implanted into the anterior chamber of the hamster eye and the ensuring vascular system was studied using scanning electron microscopy (SEM) of vascular corrosion casts. The tumor vasculature, induced mainly from the host iris vessels, showed generally disorganized and irregular patterns. Tortuous tumor capillaries of uneven contour with local dilatations and constrictions were drained by markedly dilated, thin-walled, venous vessels connected with the eye veins. Vascular sprouting and, less frequently, intussusceptive formation of new vessels were observed at the periphery and also within the tumor mass. The presence of numerous nodular outgrowths, varying in size, on the surface of dilated venules and venous vessels represent morphological evidence for continuous remodeling of the tumor vasculature. The observed features of the vascular system seem to provide a pathway for further tumor expansion. Our study showed that BHM Ma line, originating from an aggressive skin melanotic melanoma, implanted to the eye anterior chamber gave rise to rapidly growing tumors with the capability of inducing abundant vasculature which allows metastasis to the lungs, kidneys and regional lymph-nodes.

Does Melanin Affect the Low LET Radiation Response of Cloudman S91 Mouse Melanoma Cell Lines?

Melanin contains melanin-free radicals and can both absorb and produce additional free radicals and active oxygen species on exposure to various stimuli. Yet its role in the radiation responses of malignant melanoma has been little studied. In this report, three subclones of Cloudman S91 mouse melanoma clone PC1A varying in constitutive melanin content were compared with respect to killing by gamma irradiation. Radiation responses correlated with melanin content. The least melanotic line, S91/amel, was most sensitive and the most melanotic line, S91/I3, was most resistant. Curve fitting using the linear-quadratic model suggests that S91/amel is killed only by single event inactivations; S91/I3, only by double event inactivations; and S91/M1B, with intermediate melanin and radiation response, by both types of inactivations. Split dose experiments confirmed a lack of immediate split dose recovery in S91/amel and its existence in S91/I3. Potentially lethal damage and its repair could be demonstrated in both S91/amel and S91/I3. Double strand break (DSB) induction was evaluated as a function of gamma ray dose in DNA of S91/I3 and S91/amel, as well as in EMT6, a mouse mammary cancer line that lacks tyrosinase and melanin. The rates of induction were proportional to cellular melanization, i.e., the rate of DSB induction was greatest in S91/I3, least in EMT6. Levels of thioredoxin reductase (TR), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) were determined in S91/amel and S91/I3. TR was the same in both cell lines, while the other three enzymes were 3- to 4-fold lower in S91/amel.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal variation in the sensitivity of B16 melanoma to m-AMSA.

A hypothesis that m-AMSA may have greater cytotoxicity in melanin-containing tumour tissues, because it may reversibly bind to melanin, leading to prolonged drug exposure, was examined. Clonal lines of B16 melanoma which differed widely in pigmentation level were selected by isolating artificial lung colonies and in vitro soft-agar colonies, and implanting them into mice. Excision cell-survival assays performed 24 h after drug administration showed that in vivo sensitivity to m-AMSA progressively increased as pigmentation level decreased, but that m-AMSA drug levels measured 24 h after treatment were much lower in amelanotic than in melanotic lines. In dose-survival studies the reduced sensitivity of melanotic cell lines was revealed as a large shoulder (Dq = 27 mg/kg) though the terminal slopes for melanotic and amelanotic cell lines were similar (D10 approximately 31 mg/kg). Time-course studies indicated that there was no significant loss of drug from a melanotic cell line for 72 h after drug administration, though in an amelanotic cell line drug levels fell 10-fold in 10 h. There was, however, no evidence for prolonged drug cytotoxicity in the melanotic cell line. Using a fractionated drug-treatment regime, the greater cytotoxicity of m-AMSA to amelanotic tumour tissue was confirmed in a non-invasive regrowth-delay assay.

Control of pigment production in mouse melanoma cells in vitro. Evocation and maintenance.

A clonally derived amelanotic melanoma cell line repeatedly has been forced to produce pigment by the inhibitor of DNA synthesis, I-beta-D-arabinofuranosylcytosine (ara-C) at sublethal levels. One ara-C-derived melanotic line has been cloned, and has continued to produce pigment for 2 years on normal medium. The inhibitor is most effective when administered to synchronized cells in four pulses on successive days at 1.8 x 10(-5)M during the S phase of the cell cycle. Colcemid at a sublethal concentration, and growth on medium solidified with agar also evoked pigment production in this line, but a large number of other inhibitors of biosynthetic processes did not, under the conditions tested. The melanotic lines are active producers of tyrosinase (DOPA oxidase), whereas the amelanotic line produces an inhibitor of tyrosinase activity. Both enzyme and inhibitor are labile at 4 degrees C and -20 degrees C, and decay of the inhibitor in homogenates of amelanotic cells reveals a low level of residual DOPA oxidase activity. The mean population doubling time of a cloned melanotic line is 23 hr, and that of a cloned amelanotic line 16.5 hr. A similar decrease in rate of growth is found in other melanotic lines and is believed to be a significant factor in maintaining this differentiated function. Rapid growth may be related to the production of an inhibitor by the amelanotic cells.