A SEQUENCE OF VIRUS-LIKE PARTICLE FORMATION IN T H E ERGASTOPLASM OF

GREENE’S MALIGNANT MELANOMA CELLS MAKOTO TAKAHASHI, MD, AND YUTAKA MISHIMA, MD, PHD

Electron microscopic observation of Greene’s melanotic and amelanotic hamster melanoma reveals distinctive particles 90-110 mp in diameter consisting of an outer limiting membrane and a n electron dense nucleoid interior 40-50 mp in diameter, having thin spoke-like rays. The structure of these particles is es- sentially identical to that of virus-like particles found in Fortner’s melanoma but differs from mature C-type virus particles in nucleoid structure. No virus- like particles were found in the liver, kidney, or skin of non-melanoma bearing control hamsters. The sites of development of the particles are the sacs of the endoplasmic reticulum. A subcellular sequence of the development of virus- like particles within the ergastoplasm of Greene’s melanoma cells has been pro- posed. This sequence appears to begin with the bulging of the ergastoplasm and the aggregation of the ergastoplasmic ribosomes.

INCE ELECTRON MICROSCOPY OF MALIGNANT S melanoma has begun to reveal the sub- cellular organization of the malignant cells, virus-like particles have been found in the ergastoplasm of Fortner’s spontaneous mela- nomas of Syrian (golden) hamster11 and S-91 mouse melanoma.19~ 20 However, the signifi- cance of these virus-like particles in mela- noma carcinogenesis remains undetermined. For this reason, the absence or presence of such particles in the spectrum of various ma- lignant melanomas has become a subject of investigation.

Current findings have led us to examine further the cells of Greene’s melanoma, a tu- mor that has not previously been reported to contain virus particles. In the present paper, our observations are directed toward deter- mining whether virus-like particles were char- acteristic of hamster melanoma cells or whether they could be found elsewhere in hamster tissues.

MATERIALS AND METHODS Greene’s transplantable melanomas of Sy-

rian (golden) hamsters, melanotic and amel- From the Departments of Dermatology, Wayne State

University, School of Medicine, and Detroit General Hospital, Detroit, Mich. Veterans Administration Hos- pital, Allen Park, Mich., and Wakayama Medical Uni- versity, Wakayama, Japan.

Supported by Public Health Service Research Grants No. CA-08891-01 and CA-0.5580-05 from the National Cancer Institute and No. AM-07981-03 from the Na- tional Institute of Arthritis and Metabolic Diseases.

The authors wish to express their thanks to A. F. Howatson for his helpful suggestions in the preparation of this manuscript.

Received for publication June 16. 1969.

anotic, were kindly supplied by Harry S. N. Greene. These tumors were maintained by successive subcutaneous transplantation into young adult hamsters purchased from the Lakeview Hamster Colony, Lakeview, N. J. T h e transplantation was performed by the mince-suspension technique. A tumor of mel- anotic melanoma, 279A-XI, and a tumor of amelanotic melanoma, 278A-XI, both grown from the first transfer of the tumors obtained from Greene were used in this study. T h e tu- mors showed the same biologic characteristics reported by Greene.10 The excised tumor tis- sues were fixed with 6.25% glutaraldehyde in cacodylate buffer17 followed by post-fixation in 1 osmium-tetraoxide of Millonig.13 After alcohol dehydration the tissues were embedded in Maraglas 655 and sectioned on a Porter-Rlum MT2 ultramicrotome, and uranyl acetate and lead citrate stained thin sections were examined with an RCA EMU 3F electron microscope.

OBSERVATIOXS In examining the cells of Greene’s hamster

melanoma we encountered distinctive spheri- cal particles with a diameter of 90-110 mp, consisting of outer limiting membrane and a spherical, centrally located, electron dense, nucleoid structure with a diameter of 40-50 mp (Fig. 1). The nucleoid structure does not appear to have an enclosing membrane, al- though it shows some substructure and posses- ses thin spoke-like rays which may have con- tact with the outer limiting membrane (Fig. 2). Extensive examination reveals several var-

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No. 5 GREENE’S MALIGNANT MELANOMA CELLS Takahashi and Mishima 905

iations of the particles within the rough sur- within the mildly bulged ergastoplasm, ex- faced endoplasmic reticulum. Immature truding into the ergastoplasmic sacs (Figs. 3, scarcely organized nucleoid structures are seen 4). These immature central nucleoid struc-

FIG. 1 ( top) . General view of subcellular organization of Greene’s melanotic malignant mela- noma cell. V: virus-like particles, E: ergastoplasm, Q: melanosomes (X33,500).

FIG. 2 (boltom). Closer view of a virus-like particle demonstrating a limiting membrane (L) and centrally located electron dense nucleoid structure (N) with spoke-like rays (r) within the sacs of the ergastoplasm (E). Greene’s melanotic melanoma (x186,300).

906 CANCER November 1969 Vol. 24

tures closely resemble aggregates of ribosomes rounding ergastoplasm (Fig. 3). A number of and are in continuity with the regularly these nucleoid structures can be seen, regu- aligned ribosomes on the surface of the sur- larly spaced, in strands within the bulged

FIG. 3 (top). A particle bulging from the ergastoplasm. The limiting membrane (L) and nucleoid structure (N) are in direct continuation with the membranes (1) and ribosomes (n) of the ergastoplasm. Greene’s melanotic melanoma (~70,700).

FIG. 4 (bottom). Bulging stalk (S) of virus-like particle (V) from the ergastoplasm (E). Greene’s melanotic melanoma (~45,400).

No. 5 GREENE’S MALIGNANT MELANOMA CELLS Takahashi and Mislaima 907 protruded membrane (Fig. 5). In another in- a strand of nucleoid structures is narrowed stance, the protruded membrane surrounding and constricted between nucleoids (Fig. 6).

FIG. 5 (top). Multiple nucleoid structures (N) in strands are enveloped within the bulged membrane (L). Greene’s melanotic melanoma (~46,200). FIG. 6 (bottom). Nucleoid structures (N) in strands are enveloped within continuous outer

membranes (L) which show a pinched narrow (arrow) sac around each structure. Greene’s mela- notic melanoma (x62,500).

908 CANCER November 1969 VOl. 24

In some cases rows of typical mature virus- like particles consisting of limiting mem- branes, distinct nucleoid structures, and spoke-like rays are seen within sacs of the ergastoplasm (Figs. 7, 8). No virus-like par- ticles have been found in the liver, kidney, or skin of non-melanoma bearing, control hamsters.

DrscussIoN In addition to the well documented enzyme

mediated melanogenic changes occurring in malignant melanoma, there have been many recent reports of the finding of virus-like par- ticles within these tiimors.11’ 20 Accord- ing to Bernhard’s morphological typing,2 the virus-like particles of Fortner’s melanoma cor- respond most closely in structure to the C-type virus particles found in avian leucosis,lz mu- rine leukemia,3 and lymphoid leukemia of the New Zealand Black strain mice, although the described C-type virus particle lacks spoke- like rays found here.lR C-type particles have recently been reported in Burkitt’s lym- phoma.7 These particles, having a diameter of 110-1 15 mp, were enclosed by cellular mem-

brane and were present only in the cytoplasm of the infected cells. Similar particles have been reported in murine lymphoma associ- ated with reovirus type 3 infe~t i0n. l~ The particles occurring in the S-91 mouse mela- noma have been reported to belong to the classification of A-type virus particles.

The particles of S-91 mouse melanoma have been found to be formed in the rough surfaced endoplasmic reticulum. A number of authors 1.6, 9, 1 8 9 16 have also indicated that type-A virus particles appear to originate from the membrane of the rough-surfaced endoplasmic reticulum. T h e virus-like particles observed in the present study of another spontaneous hamster melanoma, Greene’s melanotic and amelanotic melanoma, have consistently ap- peared within the sacs OI the rough-surfaced endoplasmic reticulum in various stages of structural maturation. According to a recent proposal for the classification of oncogenic KNA viruses,5 C-type virus particles are sub- divided into 2 groups: the “immature C-type particle” which has an electron-lucent nucle- oid, and the “mature C-type particle” which h a s an electron-dense nucleoid. In the present

FIG. 7. The completed pinching off of the constricted limiting membrane (L) results in mature typical virus-like particles with distinct nucleoid structure (N) and spoke-like rays (r) in the endoplasmic reticulum (E). Greene’s melanotic melanoma (X78,lOO).

No. 5 GREENE’S MALIGNANT MELANOMA CELLS - Takahashi and Mishima

FIGS. 8 4 , B. Multi- ple isolated virus-like particles (V) within the ergastoplasm (E) of Greene’s amelanotic melanoma cells. x31,- 900 (A) and ~22,800 (B)

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study, no particles were found having electron- lucent nucleoids. The particles observed were essentially identical in appearance to the par- ticles described in Fortner’s melanoma.11 How- ever, the particles of both Fortner’s and Greene’s melanomas present projections radi- ating from the central nucleoid structure and, thus, differ slightly from the mature C-type particle described in the classification. Throughout the study, the formation of virus- like particles by budding of the membrane has been found to occur only from the endo- plasmic reticulum. The correlation of the bulging of the ergastoplasm at various stages, and the process of maturation of the interior of the particles have led us to the following interpretation of the sequence of virus for- mation in hamster melanoma cells (Fig. 9). In addition to the illustrated sequence, Steps I to V, in Fig. 9, it may be noted that the ribo- somes on the surface of the ergastoplasm are continuous with the aggregates of ribosome- like particles within the limiting membrane, suggesting that the nucleoid structure of the particles arises from the ergastoplasmic ri- bosomes and that the limiting membrane of the particles arises from the bulging mem-

FIG. 9. Proposed se- quence in the forma- tion of virus-like par- ticles by the bulging process of the ergastp- plasm in Greene’s mel- anoma cells.

brane of the endoplasmic reticulum (Figs. 3, 4). Dalton et al. have observed segmentation of, and occasional budding at the ends of, cylindrical forms of particles of the Moloney agent.4 These findings may correlate with the later stages of the mechanism of viral synthesis proposed in the present study.

In contrast to the persistent occurrence of virus-like particles in hamster melanoma, no virus-like particles have been found in the liver, kidney, or skin of non-melanoma bear- ing control hamsters. These findings, together with the recent reports that malignant mela- noma of Greene can be induced by the super- natant cell free fraction of homogenized tu- mors, indicate the necessity of further investi- gation on the biologic significance of these particles in malignant pigment cell growth.

Addendum: After the completion of this paper, the presence of C-type virus-like par- ticles has also been observed by electron mi- croscopy in both B-16 and Harding-Passey mouse melanomas (Novikoff, A. B., Albala, A., and Biempica, L.: Ultrastructural and cytochemical observations on B-16 and Hard- ing-Passey mouse melanomas. J . Histochem. Cytochem. 16: 299-319,1968).

No. 5 GREENE’S MALIGNANT MELANOMA CELLS - Takahashi and Mishima

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