Amelanotic Tumors Research Articles

Interference With Tumor Cell-Induced Degradation of Endothelial Matrix on the Antimetastatic Action of Nafazatrom2

The antithrombotic compound nafazatrom was evaluated in several in vivo and in vitro assays to elucidate the mechanism of its antimetastatic activity. C57BL/6 mice bearing B16 amelanotic subcutaneous tumors treated with 100 mg nafazatrom/kg/day exhibited a sixfold reduction in metastatic pulmonary lesions compared to lesion numbers in controls. The reduction in metastatic lesions was not accompanied by changes in primary tumor growth, and up to 1 microgram nafazatrom/ml did not inhibit tumor cell proliferation in vitro. Treatment of C57BL/6 mice with nafazatrom prior to iv inoculation of tumor cells failed to inhibit lung colony formation. In vitro exposure of exponentially growing B16 amelanotic cells to nafazatrom (1 microgram/ml for 72 hr) in culture did not change their ability to adhere to endothelial cell monolayers. B16 amelanotic cells degraded the matrix material of bovine endothelial cell monolayers; a heparin sulfate proteoglycan appeared to be the predominant matrix component released by these tumor cells, as judged by resistance to chondroitin ABC lyase and sensitivity to heparitinase and pronase degradation. Nafazatrom (1 microgram/ml for 72 hr) inhibited the solubilization of matrix components by approximately 60%. Tumor cell degradation of matrix components is an important event in the pathogenesis of metastasis. Thus the interference with this process appears to provide an explanation for the inhibition of malignant cell dissemination in vivo by nafazatrom.

Malignant melanoma: analysis by DNA flow cytometry

The DNA content of 14 primary and 111 secondary melanomas was determined by flow cytometry. Aneuploidy was detected in 67% of samples. The frequency with which aneuploid cells were found was similar in primary and metastatic melanomas and, in the metastatic group, for melanotic and amelanotic tumours. Aneuploid diversity was marked with a wide variation in DNA content between tumours. Serial biopsies were performed in 14 patients, and in 10 there was discordance in DNA profiles between first and subsequent biopsies. Tumour biopsies taken from different sites at the same time also showed discordance in 3 of 5 cases. These features highlight the degree of cellular heterogeneity in malignant melanoma.

Effect of theophyllin and isoproterenol on cAMP level in melanotic and amelanotic hamster melanomas.

The level of cAMP was investigated in the following three types of hamster malignant melanomas: amelanotic, depigmented and melanotic. The amelanotic and depigmented tumors were undifferentiated, with high proliferative activity, and lacked tyrosinase. The melanotic one was slow growing, highly differentiated, and expressed tyrosinase activity. Among the melanomas investigated, the higher concentration of cAMP was found in undifferentiated tumors. The single treatment of the tumor-bearing animals with theophyllin or isoproterenol did not change the cAMP level in melanotic tumors, but significantly enhanced the cAMP content in amelanotic and especially depigmented melanomas. Multiple theophyllin treatment of tumor-bearing animals caused elevation of cAMP content in all tumors, but this effect was accompanied by enhancement of tyrosinase activity only in melanotic melanoma.

Diagnostic electron microscopy for amelanotic melanoma: correlation of patient biopsy, soft agar assay, and xenograft.

In an attempt to diagnose a suspected amelanotic melanoma tumour, we examined a variety of tissue and cell samples from one patient at the ultrastructural level, which consisted of single cell suspensions of tumour cells with and without DOPA treatment, tumour cells after culture in agar with and without DOPA treatment, and single tumour cells hetero-transplanted into a nude mouse. Premalanosomes were not observed in sections of the amelanotic tumour with routine electron microscopy. Osmophilic-dense bodies, suggestive of melanosomes, were noted in the single cells in suspension treated with DOPA and cells grown in agar without DOPA treatment. Definitive premelanosomes, with an identifiable striated matrix, were only observed in cells grown into colonies in agar and treated with DOPA. Positive L-DOPA reaction products were noted in the golgi complex, endoplasmic reticulum closely related to the golgi (GERL), and in vacuoles from cells grown in agar. As controls, Cloudman S91 53.1 melanoma cells were evaluated as single cells in suspension or as colonies after culture in agar, both with and without DOPA treatment. Premelanosomes were always observed in this established melanoma cell line while DOPA-treated cells contained positive L-DOPA reaction products. The overall findings identified the tumour as amelanotic melanoma and indicated that both DOPA treatment and culture in agar were needed for the demonstration of premelanosomes.

Trace element inhibition of carcinogenesis and angiogenesis

In separate experimental models the effects of selenium, zinc, potassium, and copper on angiogenesis and carcinogenesis are compared. Inhibitory effects of Se as Na2SeO3 and of chloride salts of K and Zn, but not of Cu, on vascularization induced by amelanotic tumor implants (A Mel-4B32) in the Syrian hamster cheek pouch membrane are reported. In an earlier study the induction of new vasculature, angiogenesis, in control chambers implanted with A Mel-4B32 was initially observed on day 4 after implant. Addition of 5, 10, and 50 μg Se at the time of tumor implant delayed the initial capillary proliferation to days 7, 9, 10, respectively (Cancer Lett. 9, 353, 1980). Similarly, in this report addition of 50 μg Zn or 50 μg K delayed angiogenesis to days 7.and 6, respectively. By contrast, addition of 50 μg Cu caused severe inflammation and necrosis of the membrane before capillary proliferation could be observed and the animals had to be sacrificed.In an earlier study we reported the colon tumor incidence in 1,2-dimethylhydrazine (20 mg DMG/kg body weight for 20 weeks) -treated Sprague Dawley rats was reduced from 87 to 40% by a 4 ppm Se (as Na2SeO3) supplement in the drinking water (Cancer Lett. 2, 133, 1977). In this study rats were treated with the same dose of carcinogen, but were sacrificed 14 weeks following the last DMH injection. Drinking water supplements with 0.02% Zn (as ZnCl2), 0.5% K (as KCl), or 0.01% Cu (as CuCl2) were provided concurrently with the carcinogen and were continued until death or sacrifice. The colon tumor incidence was 18/20 in the DMH control and was reduced to 16/20 by supplemental Zn, reduced to 12/20 by supplemental K, and was unchanged (18/20) by Cu. The tumor incidences in the small intestine and Zymbal gland were all reduced by Zn, K, and Cu supplements compared with the DMH control. The hamster cheek pouch technique may provide a prescreen for potential inhibitors of chemical carcinogenesis in other experimental models.

Intermediate filaments in malignant melanomas. Identification and use as marker in surgical pathology.

Intermediate-sized filaments have been studied in human malignant melanomas and in normal melanocytes by immunofluorescence microscopy with antibodies directed against keratin, vimentin, desmin, neurofilament protein, and glial filament protein. Both human melanotic and amelanotic tumor cells and tumor metastases as well as normal melanocytes in human skin and in the rat eye contain exclusively intermediate filaments of the vimentin type. No reaction was seen with antibodies to keratin, desmin, neurofilaments, or glial filaments. These latter four antisera, however, gave strong reactions in epidermis and other epithelial tissues, muscle, or neural tissues, respectively. The results favor a mesenchymal character of melanocytes, although a neuroectodermal origin in an early developmental stage is possible. The finding that melanomas contain exclusively vimentin intermediate filaments may prove useful in differential diagnosis of melanomas from other tumor types.

Induction of pigmentation by continuous X-irradiation of amelanotic tumors of B16-XI mouse melanoma and induced change in chromosomes of amelanotic cells.

Non-pigmented tumor cells of B16-XI mouse melanoma were found to contain a diploid number of chromosomes similarly to those of melanotic tumors and the parental cells in tissue culture. A major difference between pigmented and non-pigmented cells was in the number of biarmed chromosomes per cell. There was no difference in growth rate between non-pigmented and pigmented tumors, but growth usually begins about 2 days earlier in the former. Pigmentation lost in the course of serial transplantation was restored by irradiating the non-pigmented tumor continuously with 2,500-3,000 rads/passage of X-rays during six transfer generations. In the course of repeated irradiations, the chromosomes changed structurally and numerically as the pigmentation of the tumor was gradually restored. The observations of tumor growth and chromosomal changes are discussed in relation to the pigmentation of B16-XI melanoma cells.

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.

Influence of selenium on vascularization in the hamster cheek pouch

The inhibitory effect of selenium (Na 2SeO 3) on the vascularization induced by amelanotic tumor implants (A Mel-4B32) in the Syrian hamster cheek pouch membrane is reported. Among control animals receiving A Mel-4B32 implants, 19 developed tumors out of 19 chambers with capillary proliferation being observed on day 4 after implant. Addition of 50 μg Se to the chamber at the time of tumor implant delayed the initial observation of capillary proliferation until day 10 after implant. In the 50 μg Se treated group, tumor-induced vascularization developed in 11 of 15 chamber implants and not in the remaining 4. Intermediate doses of 5 and 10 μg Se added to the chamber at the time of tumor implant delayed the initial observation of capillary proliferation to days 7 and 9, respectively, with tumors developing in all chambers. Neither sodium nor sulfite altered the vascular pattern. Capillary proliferation was observed on day 4 after tumor implant when either 50 μg NaCl or 50 μg S as Na 2SO 3 were added to the chamber at the time of implant. Provision of Se as a drinking water supplement also delayed the onset of capillary proliferation.

The structure of the glycocalyx of the golden hamster’s peritoneal macrophages following antigen stimulation

Electon-microscopy was used to study the glycocalyx of peritoneal macrophages from hamsters with transplanted melanotic and amelanotic tumours, and from control animals. The growth of transplantable melanomas in hamsters was found to cause a reduction in the amount of the material stainable with ruthenium red on the surface of the macrophages, as well as changes in the structure of the glycocalyx. The changes were more marked in the case of amelanotic melanomas.

Metastatic melanoma: the spectrum of ultrastructural morphology.

The fine structure of 26 metastatic melanomas, including 13 pigmented and 13 amelanotic tumors, as studied to define ultrastructural criteria for diagnosis. Several important features in addition to melanosome granules were identified. Dendritic cytoplasmic processes were seen in 25 of the 26 cases, cell clusters formed by concentric aggregates of several cells and their processes were present in 18, and microvilli were found on cell surfaces in all cases. The cytoplasm was complex, containing numerous mitochondria, Golgi systems, endoplasmic reticulum, nonspecific filaments, and microtubules. Nuclear morphology was variable. Basement membranes and cell junctions including desmosomes were often encountered, and some melanomas shared features with Schwann cells, including complex membrane interdigitations. All tumors contained melanosomes, although the classic forms were frequently difficult to identify and abnormal variant forms often predominated. Knowledge of the variant melanosome morphology and an understanding of the other fine structural features of malignant melanocytes can help identify those cases that lack classic premelanosome granules.

Retinal Astrocytoma

A 41-year-old man had a 6 x 6 x 5-disk diameter amelanotic tumor in the posterior fundus. The clinical and fluorescein angiographic appearance suggested a benign retinal vascular tumor, although amelanotic choroidal melanoma and retinoblastoma were diagnostic possibilities. An incisional 48-hour radioactive phosphorus (32P) uptake test was performed and the result showed an increased uptake over the tumor mass of 100% as compared to the control quadrants. The globe was enucleated and the pathologic diagnosis was isolated astrocytic glioma of the retina with minimal, if any, malignant potential. The highly developed vascular system of the tumor probably contributed to the false-positive test result.

Characterization of human malignant melanoma cell lines; V. Heterotransplantation in the hamster cheek pouch.

Seven continuous cell lines of human malignant melanoma were studied in terms of their in vivo growth potential in the cheeck pouch of the cortisonized golden hamster. Progressive tumor growth was noted only among the melanoma lines which were grossly pigmented (10/32 transplants). None of the three amelanotic tumor lines showed progressive growth. The growing tumors could be identified as melanoma on morphological grounds and by histochemical demonstration of melanin granules. Histology of the tumor lesions revealed evidence of a host reaction to the tumor transplants. This was confirmed by demonstration of circulating antibodies directed against the implanted human cells. Correlations between in vivo heterotransplantability and in vitro saturation density of human melanoma cells were not found in the present study.

Differences in cytochrome oxidase and mitochondrial ATPase in melanotic and amelanotic melanoma.

This paper deals with a description of the isolation and purification of mitochondria and melanosomes from Harding-Passey melanoma and mitochondria from Fortner melanoma together with a characterization of their cytochrome oxidase and membrane bound ATPase. In the melanotic tumor (Harding-Passey) the specific cytochrome oxidase activity was low in comparison to the enzymatic activity in the amelanotic tumor (Fortner) and in liver. The enzyme from the melanotic tumor could be inhibited only 30% by hydroxylamine, a specific inhibitor of the heme component of cytochrome oxidase, whereas the enzyme in the amelanotic tumor was inhibited by as much as 98%. This low hydroxylamine sensitivity concomitant with the low specific activity is due to a defective cytochrome oxidase in the melanotic melanoma. Similar results were obtained for membrane-bound ATPase. The enzyme from the amelanotic tumor was inhibited by oligomycin and also by an intrinsic inhibitor present in the mitochondria, whereas neither inhibitor had an effect on the ATPase from the melanotic tumor. From these results it may be concluded that the mitochondria from the amelanotic melanoma are functionally intact whereas the mitochondria from the melanotic melanoma are significantly altered or degenerated with respect to cytochrome oxidase and mitochondrial ATPase.

Malignant melanoma of the parotid gland

THIS is the report of a case of malignant metanoma of the parotid gIand, a very rare condition. Delafield and Prudden mention melanoma of the parotid only as a type of sarcoma that might occur. The term malignant melanoma here is considered synonymous with meIanosarcoma, meIanocarcinoma, nevocarcinoma and melanoepithehoma. Many observers consider the term meIanoma synonymous with maIignant meIanoma. Tumors of the parotid gIand are most commonly mixed tumors containing fibrous connective tissue, epithelium, cartiIage and cyIindromatous portions. Various forms of neoplasms may contain brownish or yehowish pigment, deposited in them by degeneration of the hemogIobin from extravassted bIood, and may be mistaken for meIanotic sarcomas. According to Moore about one-fourth of primary malignant meIanomas show extensive necrosis, and accurate histologic study is difficuIt. Rawson, Howard, Royster and Horn, in a review of 160 tumors of the salivary glands, stress the poor prognosis and relative radioresistance of these tumors. The majority of patients who were considered inoperabIe because of the extent of the lesion were treated by radiation. In none of these did this form of therapy appear to be of great benefit. OnIy 12 per cent of mixed tumors were painfu1 and only I per cent of those in the parotid gIand produced facial nerve palsy, whereas pain was a symptom in 48 per cent of malignant tumors, and facia1 nerve invoIvement was evident in 24 per cent of malignant tumors of the parotid. There is no discernibIe correIation between pigment content of the tumor and degree of malignancy. An amelanotic primary tumor may produce pigmented metastasis. The abiIity of the tumor or the metastatis to produce pigment does not support prognostic or cIinica1 interference. Metastasis of meIanin pigment to bone without accompanying tumor ceIIs has been noted.