Aggregation of normal and transformed cells attached to a substrate]

On the surface of the nirtocellulose membrane filter (pore size 0.3--0.5 mem), normal mouse or hamster embryo fibroblasts formed discrete cell aggregates. Behaviour of transformed fibroblast-like cells of 9 different lines was compared with that of normal cells. Cells of 3 transformed lines grew on this substratum as a uniform monolayer displaying no tendency to aggregation. The following 3 cell lines exposed a slightly "patchy" cell distribution on the 3rd--4th day of cultivation but were unable to form discrete aggregates. The remaining 3 lines did form aggregates but the dynamics of aggregation and the final aggregation pattern for two of them were abnormal. Only one of the 9 investigated transformed lines had the normal aggregation behaviour. Hence, in the course of the neoplastic evolution, cells lose their ability fo form aggregates on the filter surface. Mechanisms of cell aggregation and possible reasons of differencies in the aggregation behaviour between normal and transformed cells, are discussed.     

Adhesive specificity in normal and transformed mouse fibroblasts

Adhesive specificity was studied in normal and transformed $\text{Balb}\text{c}$ mouse fibroblasts by comparing the number of labeled cells collected from a suspension of these cells by aggregates of various cell types. Aggregates of the two malignant cells examined collected either very many cells (aggregates of SV3T3 cells) or very few cells (aggregates of 3T12 cells). In addition, the relative adhesive behavior of these two aggregate types did not vary according to the cell suspension in which they were circulated. These data make it unnecessary to assume that malignancy is always accompanied by a decrease in intercellular adhesion.The adhesive behavior of normal 3T3 cell aggregates, compared to the aggregates composed of either malignant cell type, varied according to the type of cells in the suspension. Aggregates of 3T3 cells collected an appreciable number of SV3T3 cells but few 3T12 cells. Collection of 3T3 cells by 3T3 aggregates was also low if the 3T3 cells of the suspension were harvested from confluent cultures. However, collection of 3T3 cells by 3T3 aggregates increased significantly, as compared to collection by SV3T3 and 3T12 aggregates in the same cell suspension, if the 3T3 suspension was prepared from sparse cultures.Flat-revertants of SV3T3 cells were also studied. These cells behave like nonmalignant 3T3 cells rather than like the SV3T3 cells from which they were derived.We suggest that malignancy may not be caused by decreased intercellular adhesion as compared to normal cells but, perhaps, by decreased intercellular recognition.     

Cell interactions and histogenesis in embryonic neural aggregates

Mixtures of cell suspensions from the optic lobe of 3-day-old and 6-day-old chick embryos form aggregates which show many ‘rosettes’ resulting from the invagination of peripheral ‘placodes’. However, in aggregates formed exclusively by optic lobe cells of either 3-day-old or 6-day-old embryos no rosettes are observed. By means of radioautographic studies of the combined aggregates, it was shown that rosettes are formed almost exclusively by cells from 3-day-old optic lobe, while those of 6-day-old embryos are predominantly found at the periphery of the aggregates. Cells from 3-day old optic lobe also form rosettes when cultured in combination with wing cells. Similar observations were done in aggregates formed by mixtures of 3-day-old optic lobe and 6-day-old liver cells. The cells from the optic lobe of 3-day-old embryos forming the concave surface of invaginating placodes and rosettes in the ‘combined’ aggregates appear polarized and wedge- or ‘bottle’-shaped. They are aligned in a highly ordered way, and abundant zonula adhaerentes, usually parallel to the cell major axis, are found at the cell apex where bundles of longitudinally oriented microtubules are present. The 3-day-old optic lobe cell which form the convex external surface of the ‘pure’ aggregates have a similar shape and organization, but in this situation apical cell bulges with randomly oriented microtubules interdigitate with those from neighbouring cells, and are joined by zonula adhaerentes usually perpendicular to the longitudinal axis of the cell.     

Growth and survival signalling in B16F10 melanoma cells in 3D culture

The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways.     

Transformation-induced alterations in adhesion : Binding of pre-formed cell aggregates to cell layers

The formation of intercellular adhesions by mouse 3T3 cells and their SV40-transformed derivatives is analysed by measuring the binding of pre-formed aggregates of these cells to cell layers or to a plastic substratum. The rationale for this procedure is to reduce the effects of cell dissociation on quantitative assessments of adhesive interactions. The fibroblasts within the aggregates retain the growth characteristics these cells show in monolayer culture. The proportion of aggregates binding is independent of the number of aggregates added and changes with time in a manner consistent with a first-order process, allowing the percent aggregates binding per unit time to serve as a parameter of intercellular adhesion. The rate of binding in homologous adhesive interactions is slower than in heterologous ones, binding in 3T3SV interactions is slower than in 3T3 interactions, and binding to cellular substrata is slower than to plastic. Binding of 3T3SV aggregates is readily distinguished from binding of 3T3 aggregates by the presence of a brief lag in binding rate, the formation of irregular projections from the bound aggregate, and a differential effect on binding rates of varying the temperature or of treating a single reactant with glutaraldehyde. Thus, there are quantitative and qualitative differences in the adhesive interactions of normal and transformed cells. The distinct binding properties of 3T3SV aggregates and the greater binding rates in heterologous interactions may be relevant to the invasive behavior of transformed cells in vivo.     

Development of normal and genetically dystrophic mouse muscle in tissue culture : I. Prefusion and fusion activities of muscle cells: Phase contrast and time lapse study

Dispersed cell cultures, derived from the forelimbs and hindlimbs of genetically dystrophic (dy/dy) and normal (+/+) day mouse embryos were studied with phase contrast microscopy and time lapse cinematography. The composition of the cell populations, and the prefusion and fusion activities of the cells were analysed. Forelimbs of both normal and dystrophic embryos consistently yielded fewer mononucleated cells, more fat cells and fewer myoblasts than hindlimbs, but there was no difference in the population of cells from normal and dystrophic limbs. During prefusion, myoblasts (both normal and dystrophic) exhibited (1) an apparent lack of contact inhibition of locomotion, which was in actuality an extensive movement of one myoblast under another; (2) formation of prefusion aggregates that broke up and realigned into new aggregates before fusion; (3) a special type of post-mitotic association and reassociation, not found among fibroblasts. Onset of rapid cell fusion of myoblasts occurred in a 4 to 8 h period, and was directly dependent upon initial cell concentration. No differences were found between cultures of normal and dystrophic cells in their prefusion activities or in time of onset of rapid cell fusion, when initial concentrations of cells were kept constant. The results of the present study are compared with those of other in vitro studies of dystrophic muscle.     

A Brief Contact with Early Embryonic Cells Induces the Differentiation of Embryonal Carcinoma Cells

An assay system was developed to detect a switch of mouse embryonal carcinoma (EC) cells to the pathway for normal cell differentiation after a brief contact with normal embryonic cells. The system consisted of (1) the mixed aggregation of AT805 EC cells with 8-cell stage mouse embryos, (2) the stationary culture of the mixed aggregates into blastocysts and (3) the cell culture of inner cell masses isolated from chimeric blastocysts containing EC cells at 2, 3 and 4 days after the initiaion of chimeric aggregation. The number of foci of EC cells which appeared in the cultures of inner cell masses was decreased with a length of contact of EC cells with normal embryos as the mixed aggregates. After 4 days' contact, only fibroblastic and epithelial cells appeared in most cultures of inner cell masses. Examination of isozyme markers of GPI revealed that such cell cultures consisting of nonmalignant cells contained cells of tumor origin. Thus, it was concluded that a brief exposure to the environment of normal embryos can regulate the tumor cells to differentiate into non-malignant cells. This conclusion was substantiated by comparing the pattern of protein spots of the tumor cells with that of non-malignant cells of the tumor origin by two dimensional gel electrophoresis.     

Dibutyryl cyclic AMP treatment of 3T3 and SV40 virus-transformed 3T3 cells in aggregates. Effects on mobility and cell contact ultrastructure

The random cell movement of BALB/c 3T3 and SV40 virus-transformed BALB/c 3T3 cells within homogeneous aggregates was studied by observing the degree of penetration of newly attached [3H]thymidine-labeled cells into the interior of the aggregates. The 3T3 cells penetrated into 3T3 aggregates an average of 0.89 cell diameter in 1.5 days, whereas the SV40-3T3 cells penetrated into SV40-3T3 aggregates an average of 3.20 cell diameters in the same time. Treatment of the aggregates with theophylline, theophylline plus prostaglandin E1, or theophylline plus dibutyryl cyclic AMP all decreased the penetration of the SV40-3T3 cells into SV40-3T3 aggregates (2.36, 1.22, and 0.79 cell diameters, respectively). The same treatments had little effect on 3T3 aggregates. The ultrastructure of 3T3 and SV40-3T3 cells in aggregates was examined by transmission electron microscopy. The 3T3 cells in aggregates were surrounded by microvilli and lamellipodia which were in contact with neighboring cells, whereas SV40-3T3 cells were nearly devoid of microvilli and lamellipodia and made contact at broader, less regular surface undulations. Treatment with theophylline plus dibutyryl cyclic AMP resulted in the appearance of microvilli on SV40-3T3 cells and also appeared to increase the area of intercellular contacts in both 3T3 and SV40-3T3 cells. These observations were supported for the surface cells of the aggregates by scanning electron microscopy.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

The binding of IgM to B lymphocytes: a comparison of the binding characteristics of IgM aggregates and EAB (IgM) complexes to normal and leukemic B lymphocytes

We have compared and contrasted the binding of Agg IgM and heavily sensitized EAB (IgM) complexes to the Fc mu receptor of normal and neoplastic human lymphocytes. Agg IgM binds uniformly to the entire SmIg+ B cell population yet normal lymphocytes require culture in order to achieve binding of EAB complexes to a subset of SmIg+ B cells. In blocking studies IgM complexes and IgM aggregates appear to detect the same receptor and with both reagents binding is influenced by the presence of Mg2+ but not Ca2+ and is inhibited by EDTA. The percentage of cells binding EAB was highest in normal B lymphocyte fractions enriched for C2+ cells (CRL+). EAB binding to cells in the CRL- fractions was negligible even though CRL- fractions contained cells which were SmIg+C-3. EAB bound only to neoplastic chronic lymphocytic leukemia cells (CLL) that expressed a high percentage of C+3 cells. Clones lacking a C3 receptor failed to bind EAB. Thus, the binding of EAB complexes to B lymphocytes appears to be associated principally with a subset that express a C3 receptor whereas IgM aggregates bind to the entire SmIg+ B cell population.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Balbc/3T3 cell transformation response to extracts of organic air samples as seen by their survival in aggregate form

Extracts of organic matter from samples of airborne particulate matter have been shown to possess components capable of transforming mammalian cells. This study was done to determine if Balbc/3T3 cells exposed to extracts of air samples could, unlike their normal counterparts, in the absence of a surface for attachment, divide on agar to form aggregates, and if these cells would demonstrate a dose-response phenomenon. Untreated and solvent treated control cells failed to form large aggregates and showed a decline in viable cell number over a 6-day period. Cells treated with either cyclohexane or acetone extracts of airborne particulate matter showed a dose-response increase in cell number along with the formation of progressively larger aggregates, findings similar to those seen with the positive, benzo[a]pyrene (BaP), control. Furthermore, these findings are in direct agreement with those in the simultaneously performed standard cell transformation assay which required 21 days to perform. Results show that survival in aggregate form is a rapid in vitro test system capable of detecting potentially carcinogenic activity in complex environmental mixtures.     

Specificity of cell adhesion: differences between normal and hybrid sea urchin cells.

The adhesive specificity of embryonic sea urchin cells from two species, and the two hybrid crosses between these species was examined by a cell-aggregate collection assay. Cells of normal Lytechinus or Tripneustes embryos were found to adhere to homospecific cell aggregates at a much higher rate than they would adhere to heterospecific aggregates. Hybrid cells adhered to collecting aggregates at an intermediate rate. The observed pattern of hybrid cell adhesion suggested that paternal gene products are capable of modifying cell surface adhesive sites as early as the mesenchyme blastula stage.     

Cytoskeletal organization affects cellular responses to cytochalasins: comparison of a normal line and its transformant

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistribution of vimentin in these cell types also occur when cells are allowed to respread from the rounded state in the presence of CD.     

Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells.

Pluripotent embryonal carcinoma cells can be triggered to differentiate in vitro by allowing them to form multicellular aggregates. Nullipotent embryonal carcinoma cells form aggregates, but further development is blocked. Pluripotent and nullipotent embryonal carcinoma cell lines were co-cultured to form mixed aggregates in order to determine whether a developmental signal produced by the pluripotent cell could induce the nullipotent cells to differentiate. Unlike pure pluripotent cell aggregates, aggregates from cultures initiated with a 1:1 mixture of pluripotent (PSA-1) and nullipotent (F9) cells formed endoderm but failed to differentiate further. The nullipotent cells did not produce a detectable soluble inhibitor of differentiation. A hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent cell line was used so that the fate of both nullipotent and pluripotent cells could be followed in autoradiographs of histological sections of aggregates labeled with ³H-hypoxanthine. Seven day old aggregates of pure pluripotent cell cultures contained endoderm, ectoderm and embryonal carcinoma cells. On the other hand, in 7 day old mixed cell aggregates, almost all the pluripotent cells became endoderm located on the outer surface of the aggregate. The nullipotent cells in the mixed aggregates assumed an internal position and remained embryonal carcinoma cells. Following the efficiency of plating of pluripotential cells in pure and mixed aggregates as a function of time showed that viable pluripotent embryonal carcinoma cells were lost at a 10 fold greater rate in mixed cell aggregates than in pure pluripotent cell aggregates. We conclude that nullipotent embryonal carcinoma cells in mixed aggregates with pluripotent cells exert a limitation on the ability of these pluripotent cells to differentiate.     

Specificity of cell-cell interactions in sea urchin embryos: Appearance of new cell-surface determinants at gastrulation

Studies on normal and hybrid sea urchin embryos show that, beginning at gastrulation, hybrid cells express cell-surface antigens specific to both species. The appearance of these antigens is shown to be correlated with a change in the adhesive specificity of hybrid cells: Beginning at gastrulation, hybrid cells recognize and adhere to embryonic cells of both normal genotypes. Prior to gastrulation, hybrid cells adhere to cells of the maternal genotype only. Two adhesion assays demonstrate these adhesive preferences. (i) When cell aggregates are placed together in a dish, Lytechnius aggregates fuse together, and Tripneustes aggregates fuse together, but aggregates of the two species do not fuse with each other. Hybrid cell aggregates, if they are past the beginning of gastrulation, fuse to both Tripneustes and Lytechinus aggregates. (ii) In a collection assay, midgastrula cells of the hybrid embryos are collected at a high rate to aggregates of either species. Pregastrula hybrid cells collect at a high rate to aggregates of the maternal species only. This change in adhesive preference is temporally correlated with the appearance of new cell surface antigens. Antiserum was prepared in rabbits against membranes from Lytechinus gastrulae. Indirect immunofluorescence tests show that hybrid cells of the cross (T♀ × L♂) express Lytechinus-specific antigens at the cell surface beginning at gastrulation. Furthermore, an apparent relationship between the new cell-surface antigens and adhesion exists in that Lytechinus cell adhesion is inhibited specifically after binding Fab fragments of the Lytechinus antiserum. The antiserum has no effect on Tripneustes adhesion. The Lytechinus adhesion-inhibiting activity can be removed by absorption of the antiserum with Lytechinus cells.     

Mobility of normal and virus-transformed cells in cellular aggregates

The mobility of embryonic chick cells and cells of four established cell lines was examined in cellular aggregates. This was done by preparing aggregates of unlabeled cells and allowing cells of the same type, but prelabeled with [3H]thymidine, to adhere to the surface of the aggregates. After 2-1/2 days in agitated liquid culture the positions of the labeled cells within the aggregates were determined by autoradiographic techniques. Since the labeled and unlabeled cells were otherwise identical, the degree of penetration of the labeled cells into the aggregates was taken as a measure of the mixing or mobility of cells in the aggregate. With this procedure, embryonic chick liver, heart, and neural retina cells were found to move an average of 2.12, 2.68, and 4.00 cell diameters inward, respectively. Mouse fibroblast BALB/c 3T3 cells moved an average of 1.13 cell diameters inward, while Simian virus 40 (SV40)-transformed BALB/c 3T3 cells moved as much as 8.80 cell diameters inward, indicating that cells of the malignant SV40-transformed line were considerably more mobile than the corresponding nonmalignant 3T3 cells. In contrast, cells of the hamster fibroblast line NIL B moved 4.17 cell diameters in 2-1/2 days, while SV40-transformed NIL B cells moved 3.00 cell diameters in the same time. It was therefore concluded that infection with oncogenic viruses does not necessarily result in increased cellular mobility.     

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro

We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/polyacryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies, within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either alpha- or beta-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.     

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro

Abstract. We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/poly-acryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies. within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either α or β-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.