neoplastic transformation in the planarian: ii. ultrastructure of malignant reticuloma

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 240:229-244 (1986)

Neoplastic Transformation in the Planarian: II. Ultrastructure of Malignant Reticuloma

FREDERICK HALL, MICHIO MORITA, AND JAY BOYD BEST Department tnf Physiology and Biophysics, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT Cadmium and phorbol ester induced tumorigenesis in the planarian, Dugesia dorotocephala, develops as a cocarcinogenic process involving initiation and promotion in the progression of neoplastic disease. Treat- ment of intact planarians with sublethal concentrations of cadmium sulfate and 12-0-tetradecanoy1phorbo1-13-acetate (TPA) induced a type of infiltrating tumor that proved to be potentially lethal. Surgical transplantation of such tumorous tissues into otherwise healthy planarians resulted in the same histopathological progression to lethality, which confirmed the metastatic nature of the neoplasia. Electron microscopic studies revealed that both the chemically-induced and the transplantation-based tumors involved, exclusively, the proliferation and differentiation of abnormal reticular cells, referred to as reticuloma cells. Reticular cells normally are ameboid, phagocytic, and are thought to provide the planarian with a phylogenetic predecessor of an im- mune surveillance system. A considerable incidence of mitosis was observed within the tumor areas; and the sequence of differentiation, from transformed stem cells to mature but nonfunctional reticuloma cells, was elucidated. This profile of differentiation supports the concept of cellular derivation via stem cell dynamics as opposed to dedifferentiation. A variety of ultrastructural abnormalities were characterized: several of which tend to substantiate the anaplastic quality of the reticuloma, while others are more specifically diagnostic for malignancy. These findings further extend the potential usefulness of the planarian malignant; reticuloma as a model system for the study of neoplastic stem cell diseases.

In previous studies, the freshwater planarian, Dugesia dorotocephala, was found to display the major phenomenology of mammalian chemical cocarcinogenesis involving initiation and promotion in the progression of neoplastic disease (Hall et al., '86). As described in the companion paper, joint expo- sure of intact planarians to a recognized mammalian carcinogen (cadmium sulfate) and a classic tumor promoter (12-0-tetradecanoylphorbol-13-acetate, TPA) resulted in the induction of an infiltrating, potentially lethal, transplantable tumor in a large pro- portion (76%) of the treated animals. These particular tumors were referred to as malignant reticulomas, an appellation which de- scribes the metastatic quality as well as the cellular components of the neoplastic disease; for both the chemically-induced and the transplantation-based tumors involved the

morbid proliferation of certain stem cells which differentiate, exclusively, into abnormal reticular cells. Histopathological characterization of the tumorigenic response to cadmium and the phorbol ester, TPA, represents the first identification of a distinct cancer cell in planaria; furthermore, the existence of this transformed, transplantable cancer cell line is suggestive of a committed stem cell in these animals. In view of the novelty of these assertions and the potential scientific interest generated by such topics, we set out to investigate the morphology and the cellular dynamics of the malignant reticuloma disease in somewhat more detail.

The freshwater planarian exhibits, throughout its lifetime, a peculiar totipotent

Address reprint requests to Jay Boyd Best.

01986 ALAN R. LISS. INC.

230 F. HALL ET AL

type of cell (neoblast), which imparts a remarkable capacity for regeneration, and without which the animal cannot long sur- vive. Yet, a degree of controversy has emerged as to whether: 1) undifferentiated neoblasts arise, during regeneration, by a process of dedifferentiation from more differentiated cell types (Steinmann, '25; Woodruff and Burnett, '65; Kido, '67; Kid0 and Kish- ida, '68; Hay, '68; Rose and Shostak, '68; Coward, '69); or 2) neoblasts are produced solely from a self-perpetuating population of stem cells that performs a crucial role in regeneration (Wolff and Dubois, '47; Dubois, '48, '49; Bronsted, '55, '69; Lender, '62; Wolff, '62; Morita et al., '69; Spiegelman and Dud- ley, '73; Morita and Best, '74, '84a,b), as well as in normal growth and continuous cellular replacement. Considered to be the only cells in the asexual planarian capable of mitotic division, neoblasts are selectively killed by certain doses of X-irradiation (Dubois, '48, '49; Wolff, '62; Lange, '68; LeMoigne, '681, a treatment that also abolishes the capability for regeneration. The neoblasts of planarians bear functional resemblances to both the stem cell systems of adult mammals and to the cells of early stage embryos; thus, they exhibit the responses of both to mutagenic agents, i.e., tumors and teratomas (Foster, '63, '69; Best and Morita, '82; Best, '83). Since mitosis in the asexual planarian is restricted solely to neoblasts (Morita and Best, '84b), it was anticipated that these would be the only cells capable of undergoing the promotional events required for transformation into malignancy; and hence, tumorigenesis in the planarian would ultimately involve the transformation of these particular stem cells, i.e., the malignant tumors would be expected to be neoblastomas (Best and Morita, '82). While the cadmic ion alone produced only localized cellular proliferations and, subse- quently, benign tumors consisting of a het- erogeneous mixture of normal cell types, the addition of TPA to the cadmium-containing solutions (or TPA alone, to a lesser extent) produced a progressive, infiltrating, malignant type of tumor composed exclusively of reticuloma cells and their immediate precursors. The observed mitotic activity of the malignant tumors, combined with the monotypic differentiation observed in the tumor cell populations, suggests that not only is a stem cell line involved, but that a committed (transformed) stem cell line is involved in the progression of this particular neoplastic disease (Hall et al., '86).

In recent years, electron microscopy has served to make important contributions to the study of the pathogenesis of neoplastic diseases. Reports that an increasing variety of ultrastructural abnormalities are characteristic of, or are correlated with, the neoplastic state (Bernhard, '69; Ghadially, '75, '80, '82; Gyorkey et al., '75; Trump et al., '78) has expanded the field of diagnostic electron microscopy to clinical advantage (Rosai and Rodriguez, '68; Bonikos et al., '76; Gyorkey et al., '75; Trump et al., '78; Ghadially, '80). Identifiable changes in membrane character, changes in nuclear shape and appearance, changes in cytoplasmic components and or- ganelles, and changes in intercellular connectivity are actively investigated and utilized nowadays in differential diagnoses; for the early stages of differentiation are often observable on the ultrastructural level when they cannot necessarily be recognized using routine light microscopy. Extending the insights of electron microscopy transphy- letically to the planarian system, the following study was designed to examine the planarian malignant reticuloma on the cellular and subcellular level, in order to characterize some of the ultrastructural abnormalities associated with the disease, and to determine the etiology of the reticuloma cell line.

MATERIALS AND METHODS

Asexual planarians of the species Dugesia dorotocephala were selected for normal morphology and similar lengths (18 to 20 mm) from a laboratory colony that had been main- tained in healthy condition, in aged tap water, and fed twice a week on raw beef liver. The experimental animals were exposed to solutions containing various concentrations of cadmium sulfate and the tumor-promoting phrobol ester 12-O-tetradecanoylphorbol-13- acetate (TPA). Concentrated stock solutions of TPA (Sigma Chemical Co., St. Louis, MO) were prepared in acetone at 200 pl/ml, and stored at -20°C. A weekly dilution to 0.1 pgI ml in aged tap water was carried out, and this solution was refrigerated at 5°C until final dilutions were made each day at room temperature. Stock solutions of cadmium sulfate (3CdS04:8H20, FW 769.556) were prepared in aged tap water, at a concentration of 120 mgA, and then mixed with TPA and diluted to the final concentrations. As described in the previous paper (Hall et al., '86), a concentration of 0.3 ppm 3CdS04 :8H20 (which corresponds to 0.13 ppm Cd)

PLANARIAN MALIGNANT RETICULOMA 231

combined with 0.01 ppm TPA was considered to be optimal for planarian tumorigenesis, i.e., the induction of malignant reticuloma disease. The subjects were exposed to the test solutions for a period of 2 weeks, then they were returned to aged tap water. In order to eliminate the possible promotional aspects of nutrition, the planarians were fasted during the entire experimental period. The subjects were observed closely during the latent period, which was determined to be 30 days, after which time the overt occurrence of neoplastic transformation was quantified. Some tumor-bearing animals were selected for transplantation studies, others were prepared for histological and ultrastructural examination, while the remainder became the subjects of continued observation under con- ditions of normal care and feeding.

Selected specimens were fixed for 1 h in a mixture of 1.5% glutaraldehyde and 0.5% formaldehyde adjusted to a pH of 7.3 with 0.1 M phosphate buffer solution and were then postfixed for 1.5 h in 1.0% buffered osmium tetroxide. Following fixation, the specimens were dehydrated through ethanol (from 50 to loo%), placed in propylene oxide, and then embedded in Polybed 812 resin. Sections were cut with glass or diamond knives on a Sor- vall Porter-Blum MT-2 ultramicrotome. Thick sections (1 pm) collected from each specimen block were stained with either to- luidine blue or a modified Richardson stain, and examined with a light microscope to de- lineate the tumor regions. Thin sections (800 A), cut from selected regions, were stained with uranyl acetate and lead citrate (Sato, '68). Observations were made with a Philips electron microscope, EM-200, operating at 80 kV. The electron micrographs were taken at original magnifications x 7,000 to ~20,500 and then enlarged photographically.

OBSERVATIONS

Planarian malignant reticuloma disease is a tumorous condition that is progressive, potentially lethal and transplantable (Hall et al., '86). The chemicaI cocarcinogen-induced malignant tumors originate, invariably, in the postpharyngeal mesenchyma and subse- quently spread, by infiltration, throughout the afflicted animals. The malignant tumors are characterized by a distinctive cellular composition consisting of: 1) mitotically active assemblies of (presumably transformed) stem cells; 2) mature tumor cells, called reticuloma cells, which interdigitate extensively with each other, often resulting in expansive

tissue formations; and 3) newly differentiating cells intermediate in morphology between the transformed stem cells and the mature tumor cells. The same cellular composition is consistently observed in the chemically-induced tumors, in the metastatically afflicted tissues, and in the infiltrating tumors produced by surgical transplantation.

A thorough electron microscopic examination of the tumor areas revealed the profile of differentiation of the malignant reticuloma cell, from mitotically active precursors through a sequence of intermediate stages to the distinctively transformed end product. The precursor cells are generally ovoid, displaying the principal characteristics of neoblasts, including: a relatively large nucleus, numerous free or poly-ribosomes, mitochondria, and nuclear satellite material within the meager cytoplasm. On the basis of our observations at the ultrastructural level, these precursor cells are virtually indistinguishable from normal neoblasts; however, the functional restriction of totipotency to a single cell type suggests that these stem cells are, indeed, transformed. As illustrated in Figure 1, the outset of differentiation involves a gradual transition in which the cell grows markedly in size, the nucleus to cytoplasm ratio declines, heterochromatin becomes marginated, the shape of the nucleus becomes increasingly irregular, mitochondria abound, glycogen content increases, and lysosomes appear within a fibrous cytoplasm. Nuclear satellite material persists, yet diminishes with each succeeding stage. Mitotic figures are consistently observed in the assemblies of transformed stem cells within the tumor areas but never in differentiating or mature reticuloma cells. A close examination of the mitotically active stem cells (Fig. 2b) unveils a complement of electron-dense nuclear satellite material within each dividing cell; a complement of nuclear satellite material that is similar to, but distinguishable from, the condensed chromosomes (Fig. 2a), and is almost always associated with mito- hondria.

The mature reticuloma cell (Fig. 3a) can be identified on the basis of its strikingly bi- zarre appearance and its relative paucity of cytoplasmic structures. Displaying nuclei of extremely irregular shape, these prominent cells interdigitate extensively with neighbor- ing cells, forming masses that constitute a large portion of the tumor volume. As unusual as is the gross appearance and as sparse the cytoplasmic constituents, there are

232 F. HALL ET AL.

Fig, 1. Electron micrograph montage of planarian malignant reticuloma cells depicting the sequence of development: from newly differentiating, presumably transformed, cells (RT) displaying nuclear satellite material NSM), through an intermediate stage (RM-cen-

ter), to the mature cancer cell (RM-bottom) displaying an irregular nucleus (N) and expansive cytoplasm containing glycogen granules (g), mitochondria (m), and lysosomes Cy). Horizontal bar = 1 fim. ~11,200.


Fig. 2. Electron micrograph montage of mitotic cells tiating cells, and nuclear satellite material (NSM) is within a malignant tumor (b). The ultrastructural fea- present in each cell. This nuclear satellite material, as tures displayed by the cell in metaphase (MU, the cell in shown in the insert (a) which depicts another trans- telophase ( M Z ) , and the newly differentiating cells at the formed cell in metaphase, is distinct from the condensed bottom of the plate are indistinguishable from those of chromosomes (Chr) and appears to be closely associated neoblasts: The diminutive cytoplasm contains numerous with mitochondria (m). Horizontal bar = 1 pm. free or poly-ribosomes (R), a prominent nucleolus (Ne) is a: X 17,600; b: X 11,200. observable within the nucleus (N) of the newly differen-

234 F. HALL ET AL.

Fig. 3. a) Electron micrograph of a typical reticuloma cell (RM) displaying a bizarrely shaped nucleus (N) containing aggregates of heterochromatin, some of which is compressed along the nuclear membrane. The cytoplasm contains an abundance of glycogen granules (g), mitochondria (m), and some lysosomes (Ly), yet appears to be devoid of granuIar endoplasmic reticulum. HorizontaI bar = 1 pm. x 12,600. b) Electron micrograph of a normal (control) reticular cell (RC) showing nuclear pockets

but not the deep invaginations that pervade the cancer cells. Heterochromatin is characteristically marginated. Glycogen, mitochondria, and lysosomes are present in the cytoplasm in considerable quantities; however, granular endoplasmic reticulum (Er) is well developed and phagosomes are often observed in the normal, functional reticular ceii. Lipid is labeled (L). HorizontaI bar = 1 pm. ~19,500.


certain ultrastructural features that betray the lineage of the planarian cancer cell: Het- erochromatin is similarly marginated along the inner nuclear membrane in both the normal reticular cell (Fig. 3b) and in the malignant reticuloma cell, and glycogen granules as well as mitochondria are abundant. Pri- mary lysosomes are characteristic of both; however, phagosomes and secondary lysosomes, typical of the normal reticular cell, are not observed in the reticuloma cells. Granular endoplasmic reticulum, prominent in the normal reticular cell, is not well developed in the malignant state. The presence of distinctive subcellular similarities and components, yet to an apparently less functional degree, indicate the malignant reticuloma cell to be an anaplastic version of the planarian reticular cell.

Nuclear satellite material, alias nuclear emissions, alias chromatoid bodies, is often considered to be the hallmark of planarian stem cells (LeMoigne, '66, '77; LeMoigne et al., '66; Sauzin, '66, '68; Morita, '67; Morita et al., '69; Morita and Best, '74, '84a,b; Cow- ard, '74; Hay and Coward, '75). Nuclear satellite material is not only abundant in the transformed stem cells but remains conspicuous in differentiating and often in mature, reticuloma cells as well. Indeed, nuclear satellite material is produced in comparatively exaggerated amounts by these transformed stem cells, often forming unusual "globular clusters" in the perinuclear cytoplasm. Copi- ous amounts of this electron-dense granulo- fibrillar material are observed in the perinuclear cytoplasm of differentiating reticuloma cells (Fig. 4a), often within the deep- ened nuclear invaginations. The nuclear satellite material was found to diminish with differentiation (Fig. 4b) and is observed no- ticeably further from the nuclear membrane as the shapes of the interdigitating cells become more irregular; decreasing, eventually, to persist only as vestiges in the latter stages. A close, often intimate relationship exists between mitochondria and nuclear satellite material in early stage reticuloma cells (Fig. 4c), as well as in normal neoblasts: a relationship which is examined in further detail in an upcoming paper (for abstract, see Morita et al., '85).

Intracellular membrane ensembles, known as annulate lamellae (Swift, '56), are consistently observed in the expansive cytoplasm of reticuloma cells (Fig. 5a). This cytomem- brane system, composed of parallel double

membranes containing numerous pores, or annuli, appears either as stacks or as concentric circles, depending on the section angle. Juxtanuclear annulate lamellae are often found in the early stages of reticuloma cell differentiation (Fig. 5b), then in the more peripheral cytoplasm during the later stages (Fig. 512). These structures, considered to be transitory in normal cytodifferentiation, are often seen in apparently mature reticuloma cells.

Nuclear and plasma membranes take on an increasingly irregular appearance in the more differentiated states of the reticuloma cells. Often beset with deep invaginations (Fig. 6a), the nucleus eventually displays an aberrant geometry that exceeds the range of variation of normal cells. The plasma membrane also develops from the generally ovoid shape, as seen in thin sections, to exhibit filopodial extensions which tend to form elab- orate interdigitations with adjacent reticuloma cells. The quality of the cytoplasm itself becomes increasingly fibrous and electron dense during the process of differentiation. An examination of the interdigitations under higher magnification (Fig. 6b) reveals ,the presence of fibrous "junctions" arrayed along the contacting plasma membranes. These contacts, similar to what is referred to as intermediate junctions or zonulae adhaerens (Ghadially, '75, '82; Threadgold, '761, are formed at points where cytoplasmic fibers converge and condense along the membrane.

The arrows in Figure 7a point to the concentric rings of an abnormal cytoplasmic complex observed in a malignant reticuloma cell. This membranous whorl is distinct from both the annulate lamellae mentioned earlier and the glycogen body described else- where (Ghadially, '75, '82; Saito et al., '84). First described erroneously as virus-like particles (Zucker-Franklin, '63), and later more correctly as ribosome-lamella complexes (Ka- tayama et al., '72) or granulolamellar structures (Daniel and Flandrin, '74), these ribosome-studded membrane arrays are found in certain neoplastic diseases, particu- larly the leukemias (Ghadially, '80). Ribo- some-studded lamellar whorls were observed in only a few malignant reticuloma cells, yet never in normal cells. Although the observed incidence in planarian malignant reticuloma cells is admittedly low, the presence of this cytopathological "marker" in even a few transformed planarian cells is remarkable. At this point, it is appropriate to emphasize

236 F. HALL ET AL.

Fig. 4. a-c) A pervasive feature of differentiating re- As the process of differentiation continues (b), this nu- ticuloma cells is the massive display of nuclear satellite clear satellite material diminishes to vestiges which ap- material (NSM) during the early stages. Copious pear with increasing distance from the nucleus (N). A amounts of this electron dense material is observable in close, often intimate, association between globular clus- the perinuclear cytoplasm, often forming globular com- ters of nuclear satellite material and mitochondria (m) plexes as shown in the cell in a. This cell is beset with can be seen under the higher magnification of c. Horizon- nuclear invaginations, yet still displays the centromere tal bar = 1 pm. a: ~20,800; b: x 13,700; c: ~ 3 0 , 8 0 0 . (Ctm) and associated remnants of its spindle apparatus.


Fig. 5. a-c) Annulate lamellae (AL) are consistently observed in the cytoplasm of malignant reticuloma cells (a) along with glycogen granules (g), mitochondria (m), and nuclear satellite material (NSM). These concentric intracytoplasmic structures are found in close proximity to the nucleus (N) of newly differentiating cells (b); they

then appear consecutively further removed, as the volume of the cytoplasm and the extensiveness of the plasma membrane increases. A Golgi apparatus is observable along with the annulate lamellae in c which represents a higher magnification of a. Horizontal bar = 1 pm. a: X7,400; b: x 11,400; c: x 16,300.

Fig. 6. a,b) Membrane irregularities characterize the appearance of mature but nonfunctional, reticuloma cells: The cell shown in the upper electron micrograph (a) displays an extremely irregular nucleus (N) beset with intense invaginations. Glycogen granules (gl, mitochondria (m), remnants of nuclear satellite material (NSM), and lysosomes &y) are seen in the cytoplasm, but no phagosomes are present in this malignant cell. Extensive interdigitations of the plasma membrane with

those of other reticuloma cells is a distinguishing feature of the tissue formations involving these cells. The large arrows point out a section of the membranous interdigitations that are viewed under higher magnification in b. Note the generally fibrous character of the cytoplasm. These fibers appear to condense at various points along the plasma membrane (arrows). Horizontal bar = 1 pm. a: x 11,200; b: x 32,800.

Fig. 7. a) Electron micrograph of a planarian reticuloma cell (RM) displaying an unusual ribosome-lamella complex (arrows) that is morphologically distinct from the annulate lamellae. This type of structure has not been observed in normal planarian cells; yet, remarka- bly, similar concentric lamellar bodies have been associated with a variety of mammalian neoplasms, including the leukemias. A newly differentiating, presumably transformed, reticuloma cell (RC) containing lipid (L) is

also shown. Horizontal bar = 2 pm. ~8,880. b) Electron micrograph showing an apparently normal reticular cell (RC) with an ovoid nucleus (N) and numerous lysosomes surrounded by and invaded by the more electron dense cytoplasm of reticuloma cells (RM). The arrows point out the cytoplasmic extensions, or filopodia, which depict the infiltration on a cellular level. Horizontal bar = 2 pm. ~ 6 , 9 0 0 .

240 F. HALL ET AL.

that the ribosome-lamella complex is defi- nitely not a viral particle as was originally suggested (Zucker-Franklin, '63); nor were any virus particles or inclusions ever observed in either the nucleus or the cytoplasm of the chemically transformed malignant reticuloma cells.

As mentioned earlier, the malignant tumors in the planarian were found to be comprised solely of reticuloma cells and their immediate (transformed) progenitors, with reticuloma cells forming interdigitations with adjacent cells in the process of differentiation, growth, and maturation. While the well-defined tumor areas are indeed found to be quite uniform in cellular lineage and composition, these transplantable tumors are in- vasive: they do metastasize, spreading by a process of infiltration into more remote regions and eventually throughout the entire animal. The invasion of normal tissues is depicted on the cellular level in Figure 7b, in which the cytoplasm of a group of apparently normal cells is seen being encroached upon by the cytoplasmic extensions, or filopodia, of the aggressive tumor cells. Both the normal neoblast and the normal reticular cell are known to be migratory: both capable of travel by ameboid movement to distant sites within the animal. Likewise, the transformed stem cells and the early stage reticuloma cells exhibit an ameboid appearance; however, the mature reticuloma cells, with their extensive cytoplasmic interdigitations, are considered t o be relatively immobile. It appears that the ability to divide is lost at the outset, and the ability to migrate considerable distances is lost during the later stages of cytodifferentiation. Thus, the observed metastatic spread of the malignant reticuloma disease presumably involves not only the translocation of newly differentiating reticuloma cells but includes the migration, or transplantation, of mitotically active precursors as well.

DISCUSSION

The imperative to explore the processes of carcinogenesis in simplified, yet biologically relevant, model systems remains as one of the foremost challenges of basic science. In this regard, the demonstration that the freshwater planarian responds to mammalian chemical cocarcinogens with tumorigenesis should be of considerable interest. In the accompanying companion paper (Hall et al., '861, we reported that cadmium and phorbol

ester induced tumorigenesis in Dugesia d e rotocephala exhibits the major phenomenology of mammalian cocarcinogenesis involving initiation and promotion in the progression of neoplastic disease; and that a certain type of infiltrating tumor, which is transplantable and potentially lethal, was induced. Of particular interest was the finding that the tumorous tissues were not only mitotically active but were comprised of tumor cells whose differentiation was limited to a single type of cell: a cancerous derivative of the reticular cell. Since the characterization of this malignant reticuloma cell represents the first identification of a planarian cancer cell that is distinct from the much-studied neoblast, electron microscopy was employed to investigate the origin, the differentiation, and the cytopathology of this novel cell type. The results of these studies tend to bear di- rectly on several important aspects of the developmental biology of this animal, as well as on the processes of neoplastic transformation and tumorigenesis.

The differentiation of the planarian cancer cell is clearly displayed within the tumor areas as a progression of intermediate stages, from mitotically active neoblastlike precursors to the profoundly abnormal reticuloma cells. The developmental progression of cellular phenotype involves gradual changes in both nuclear and cytoplasmic character which parallel the growth and the maturation of the malignant cells. The ephemeral production of nuclear satellite material, a distinctive cytoplasmic marker whose transitory occurrence has been used to elucidate the origin and differentiation of blastemal cells (Morita and Best, '84b), of epidermal cells (Morita and Best, '74), and of muscle cells (Morita and Best, '84a) during regeneration, provides additional reinforcement for this conclusion. Produced in massive quantities by the transformed stem cells, the nuclear satellite material diminishes and migrates further from the nuclear envelope with each succeeding stage of differentiation, eventually to disappear or remain as mere vestiges in the latter stages. The presence of nuclear satellite material in these mitotically active cells indicates that they are, indeed, stem cells prior to cell division. There is a conspicuous lack of evidence for cellular dedifferentiation in either the tumor areas or in the surrounding tissues. Thus, the results of this study serve to emphasize the role of stem cells in planarian neoplasia; however, a


new and potentially important dimension is necessarily added. The generation of a single differentiated cell type within both the chemically induced and the transplantation-based tumors suggests that the phenotypic potential of the cocarcinogen-treated neoblasts has become restricted. More specifically, during the process of neoplastic transformation, the stem cells have become committed prior to the loss of mitotic competency. The concept of totipotency regarding planarian stem cells must, heretofore, be modified to include uni- potency in the case of neoplasia. These committed (transformed) stem cells are presumably responsible for maintaining the transplantable population of mitotically active precursors within the infiltrating tumors as well as serving as progenitors for the distinctive reticuloma cells.

As discussed in the accompanying paper (Hall et al., '86), there are several functional and operational similarities between the malignant reticuloma disease of the planarian and the leukemias of the higher animals, including the specific leukemogenic action of the phorboid tumor promoter (Berenblum and Lonai, '70; Armuth and Berenblum, '74) when it is administered alone. In addition, TPA is reported to stimulate cellular proliferation in lymphoblasts (Whitfield et al., '73) and lymphocytes (Estensen et al., '78; Mastro and Pepin, '821, and to modify cellular differentiation in myeloid leukemia cells (Hozumi et al., '82; Lotem and Sachs, '82) and erythroleukemia cells (Rovera et al., '77; Diamond et al., '78; Yamasaki et al., '77, '82). While these operational similarities are intriguing, it is on the ultrastructural level, as revealed by electron microscopy, that more convincing parallels become evident, and the biological correlations with mammalian hematopoietic stem cell diseases begin to emerge. Ancillary to the general histological observations of invasion, metastasis, and an elevated incidence of mitosis, there are abundant ultrastructural characteristics that appear to be correlated with malignancy in higher animals (Bernhard, '69; Ghadially, '75, '80, '82; Gyorkey et al., '75; Trump et al., '78). Among the multitude of representative characteristics, an irregularity of nuclear shape, nucleo- lar margination, a general lack of cytoplasmic specialization, a comparative decrease in rough endoplasmic reticulum, the presence of annulate lamellae, changes in plasma membrane character, and alterations in intercellular connectivity all find relevance in

the study of the planarian malignant reticuloma.

Commonly featured in the cytoplasm of certain germ cells, in embryonic cells, in newly differentiating cells, and in tumor cells (Kessel, '68; Witschnitzer, '70), annulate lamellae bear some resemblance to nuclear pore complexes Waul, '77) and are generally thought to originate from the nuclear envelope. Annulate lamellae, as seen in malignant reticuloma cells, have also been reported in the cytoplasm of differentiating neoblasts (Morita et al., '69) and in the gastrodermal cells of regenerating, but not in intact, planarians (Coward, '79). Considered to participate in the elaboration of intracellular membrane arrays, the specific function of the annulate lamellae remains obscure; yet the transitory presence in differentiating cells implies an important role in this process: a role which is possibly exaggerated by the increased demands of the malignant state.

The existence of various intercellular con- nections, or alterations thereof, are relevant to the study of neoplasia and are often used for diagnostic purposes (Rosai and Rodriguez, '68; Martinez-Palomo, '70; Bonikos, et al., '76; Gyorkey et al., '75; Trump et al., '78; Ghadi- ally, '75, '80, '82). Junctions of the "intermediate" type are found in embryonic connective tissue cells (Ross and Greenlee, '66) and in certain chemically-induced tumors of mesenchymal origin (Clarke, '70). Structures similar to intermediate junctions were observed between interdigitating planarian reticuloma cells, whereas only gap junctions are commonly observed between normal reticular cells (unpublished observations); however, the relative occurrence and the functional significance of these curious cell contacts remain to be investigated in further detail.

Whether investigating mammalian or tur- bellarian neoplasms, one should bear in mind, nonetheless, that many of these de- scriptive characteristics, such as the presence of annulate lamellae, a marginated nucleolus, or poorly developed rough endoplasmic reticulum, are exhibited by normal differentiating cells during normal development; they are, therefore, more indicative of the undifferentiated (anaplastic) status of the cells rather than representative of actual cytopathology. One should also bear in mind that the anaplastic quality of mammalian tumors is generally considered to be the re-

242 F. HALL ET AL.

sult of pathological constraints on differentiation rather than actual dedifferentiation of mature cells (Widman, '78). Several of the observed ultrastructural features of the malignant reticuloma cells such as glycogen granules, lysosomes, and distinctively marginated heterochromatin betray its descent from the reticular cell line; while others such as prominent nucleoli, nuclear satellite material, sparse granular endoplasmic reticulum, the presence of annulate lamellae, and the lack of phagosomes indicate an anaplastic status and diminished function.

Of the various ultrastructural features described in planarian malignant reticuloma cells, the extreme irregularity of nuclear shape and the occurrence of abnormal ribosome-lamella complexes are more specifically correlated with malignancy (Bernhard, '69; Ghadially, '75, '80, '82; Trump et al., '78): the nuclear shape, with the degree of malignancy in general; and the ribosome- lamella complex, with the leukemias in particular. First described in chronic lymphocytic leukemia (Zucker-Franklin, '63), these concentric ribosome-lamella complexes, which are considered to be derived from rough endoplasmic reticulum (Anday et al., '73; Daniel and Flandrin, '74; Brunning and Parkin, '75; Ghadially, '821, are frequently observed in leukemic reticuloendotheliosis and are relatively rare in other neoplastic diseases (Katayama et al., '72). Now, leukemic reticuloendotheliosis, or hairy cell leukemia, is a neoplastic disorder of lympho- reticular cells characterized by anemia, leu- kopenia, and the presence of neoplastic reticulum cells (hairy cells) in the peripheral blood, the bone marrow, and the spleen; however, the nature and classification of the leukemic hairy cell, with its fine pseudopodial projections and its ribosome-lamella complexes, is the focus of much uncertainty and debate at present. Based on a wide spectrum of clinical, cytological, and histological evidence (Ghadially and Skinnider, '72; Katay- ama et al., '72; Daniel and Flandrin, '74; Haegert et al., '74; Schnitzer and Kass, '741, the origin of the hairy cells has been her- alded as either lymphocytic, monocytic, or histiocytic at one time or another; and several novel cell types, such as "flagellated cells" and "dendritic cells" (Daniel and Flan- drin, '741, have been included in the discus- sions. The totally unexpected observation, in hairy cell lukemia, of abnormal cytoplasmic projections involving red blood cells and cells of the erythropoietic series led investigators

to suggest, albeit casually, that the neoplastic transformation occurs in some primitive hematopoietic reticulum cell and that various altered cell lines are differentiated from this transformed stem cell (Ghadially and Skinnider, '72). At this point, it is tempting to speculate that the lowly planarian just might provide a degree of phylogenetic in- sight into this ontogenetic mystery.

ACKNOWLEDGMENTS

This work was supported in part by Science and Education grants from the U.S. Depart- ment of Agriculture to the Colorado State University Experiment Station.

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