Neuroblastoma: The E. coli of neurobiology?

Pergamon Press

Life Sciences, Vol . 16, pp .1649-1658 Printed in U.S .A .

MINIREVIEW

NEUROBLASTOMA:

THE E . COLI OF NEUROBIOLOGY?

Susan C . Haffke and Nicholas A. Seeds Department of Biophysics and Genetics University of Colorado Medical Center Denver, Colorado B0220

Since the first reports (1,2,3) in 1969 of the establishment of cell lines from the mouse neuroblastoma 01300, there has been a multitude of research performed with these cells.

It is doubtful that any other research

tool has had a greater impact on recent neurobiological investigations . Many pharmacologists and neurochemists as well as cell biologists were quick to realize the advantage of such a homogenous self-propagating experimental system that possessed morphological, biochemical and electrical properties of mature neurons .

Obviously, we cannot discuss all these studies in this

review (see ref 4,5 for more extensive review), however, we'll try to summarize the results and determine the contributions that this now famous tumor cell has made to neurobiology . Neuroblastoma tumor C1300 arose as a spontaneous tumor which has been maintained since 1940 at the Jackson laboratories by serial transplantation in strain A/J mice .

Both in subcutaneous tumors and in suspension cultures,

these cells have a round, undifferentiated neuroblast morphology .

However,

many clonal lines of neuroblastoma, when given a surface on which to attach, such as glass, collagen or commercially treated tissue culture dishes, undergo a striking change in morphology .

The most prominent characteristic

of this change is the extension from the cell body of one to four elongated cytoplasmic processes over 50 Um in length .

Since specific functions have

not been attributed to these processes, they are referred to as neurites . 1649

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Neuroblastoma

Within a few days, these processes elongate to form a complex network giving the cultures the appearance of differentiated neurons .

Furthermore, these

cells are stained by the Bodian procedure, a neuron specific stain, while those with the round neuroblast morphology are not (2) . Although most clonal lines are able to morphological differentiate soon after plating, this ability seems to vary with the clonal line being studied and the conditions of cell culture .

An example of such a clone is

neuroblastoma N18 which is interesting in that the proportion of cells with processes varies more than 100-fold depending on its environmental conditions (6) .

This clone and many others can be induced to undergo morphologi-

cal differentiation by the relatively simple procedure of lowering the serum concentration within the growth media.

The effects of serum concentration

on process formation are at least two fold .

First, factors are present in

serum that influence the attachment of cells to the plate and cause an alteration of the balance between process extension and retraction .

Thus the axon

outgrowth seen after reduction of serum concentration in the culture media is related, at least in part, to a more stable interaction between the cells and the plate .

Secondly, the presence of serum stimulates cell division,

nucleic acid and protein synthesis .

Since neuroblastoma cells retract

their processes prior to mitosis, reducing the serum concentration blocks cell division, thus allowing the uninterrupted extension of neurites . Other methods currently being employed to induce morphological differentiation include treatment of the neuroblastoma cells with bromodeoxyuridine (7), fluorodeoxyuridine (3), cytosine arabinoside (8), x-irradiation (9) and hypertonic growth media (10) .

Of possibly more biological significance

is the observation that glial cells release a factor into culture media that induces a high degree of morphological differentiation in neuroblastoma cells without affecting their growth rate (11) .

Furthermore, this

factor(s) is distinct from the popular nerve growth factor which fails to elicit morphological differentiation in clonal lines of C1300

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Vol. 16, No . 11

in contrast to normal sympathetic nerve

(12) .

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One of the most interesting

observations is the ability of dibutyryl cyclic adenosine 3', 5'-monophosphate to bring about differentiation that is largely irreversible (13,14) . Following a three day exposure to dibutyryl cyclic adenosine 3', 5'-monophosphate, many differentiated cells are still present a week or more after removal of the agent .

Agents that elevate intracellular cAMP levels such as

prostaglandin E1 (15,16) the phosphodiesterase inhibitors, Ro20-1724 and papaverine (16) are often more efficient than dibutyryl-cANP in bringing about this morphological transition .

In addition, serum withdrawal leads to

a two fold increase in intracellular cAMP levels (17) .

However an increase

in cellular cAMP levels alone is not sufficient to produce morphological differentiation in all clones (17) . Electron microscopy reveals these neuroblastoma neurites contain large numbers of microtubules and neurofilaments which are characteristic of the axon and dendrites of mature neurons (2) .

Additional studies have shown

that neuroblastoma cell homogenates contain a high level of the colchicine binding protein, tubulin (18) .

Neurite outgrowth by the neuroblastoma cells

is blocked by the presence of either colchicine or vinblastine, two antimitotic drugs known to inhibit microtubule polymerization .

However, inhibi-

tion of protein synthesis does not affect initial neurite formation (6) suggesting that initial nerve outgrowth is dependent on the polymerization of tubulin into microtubules from a pool of preformed tubulin subunits . Although the differentiated neuroblastoma often have neurites millimeters in length and contain considerably more microtubules than the undifferentiated cell form, surprisingly the cells of both morphologies contain identical amounts of tubulin subunits (19) . Another obvious factor in nerve outgrowth is the interaction between the cell surface and the substratum .

Since changes in the cell surface

represent important aspects of differentiation and morphogenesis, with surface glycoproteins and glycolipids being implicated in cell recognition

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Neuroblastoma

and contact phenomena, a variety of studies have been undertaken to compare the cell membranes of different neuroblastoma clones and those treated with agents that induce differentiation .

Glycosphingolipid profiles of cells

grown in suspension as neuroblasts or in surface cultures as neurons are identical (20) .

A comparison of trypsinates from pairs of neuroblastoma

clones that differed in ability to form neurites revealed a distinct difference in cell surface glycopeptides between cells that could and could not extend neurites (21) ; however, no difference was found between the surface glycopeptides of the differentiated and undifferentiated morphology of a single clone (22) .

The more sophisticated approach of preparing antisera

to cells with neurites and the absorbing the antisera with non-neurite cells of the same clone produced a "differentiation" specific antisera that also cross-reacted with brain tissue (23) .

Similarly, a protein of 78,000

daltons was preferentially exposed to lactoperoxidase iodination on the surface of dibutyryl cAMP differentiated cells but not on treated cells where differentiation was prevented by suspension culture (24) . Accompanying these morphological changes, neuroblastoma cells also develop some electrophysiological and biochemical characteristics of sympathetic neurons .

Intracellular recordings reveal that neuroblastoma cells

are capable of spontaneous and repetitive depolarization5in addition to generating action potentials in response to electrical stimulation (26) . Some cells which exhibit well-developed action potentials .upon electrical stimulation also show changes in the action potential upon application of acetylcholine (27,28) .

Of these cells, one-half were insensitive to acetyl-

choline ionophoresed along one of their processes, suggesting an analogy with normal axons and dendrites .

This acetylcholine receptor is nicotinic

in nature and binds a-toxin of Naja ni$ricollis (29) .

Furthermore, some

neuroblastoma cells are electrically coupled via their processes (27) . A marked increase in electrical excitability was found in clone N18 concomitant with neurite formation, while clone NlA-103 which is unable to form

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Neuroblastoma

neurites lacks electrical excitability (30) .

However, cells need not have

neurites to generate an action potential (26) . A trophic interaction similar to early synapse formation occurs between neuroblastoma cells and clonal skeletal muscle cells in culture (31) .

An

increased chemosensitivity to acetylcholine arises where the neuroblastoma neurite comes in contact with the fused muscle cells .

However, to the

chagrin of many, no chemically mediated synaptic transmission has been found even when the neuroblastoma cells have been confronted with heart, vas deferens or other suitable target tissues . Several hundred clones and subclones of the neuroblastoma have been isolated thus far in a half dozen different laboratories .

These clones show

considerable variation in their morphological, electrical and biochemical properties .

Biochemically, neuroblastoma clones can be divided into four

major categories with respect to neurotransmitter synthesis ; strictly cholinergic, containing high levels of choline acetyl-transferase ; strictly adrenergic, containing high levels of tyrosine hydroxylase ; clones that synthesize neither acetylcholine or catecholamines (32), and interestingly, clones that synthesize both types of neurotransmitters . (33) .

Furthermore,

all clonal lines observed thus far contain acetylcholinesterase .

A summary

of same of these biochemical activities and their inducibility in cell culture is given in Table I .

A variety of treatments, serum removal (34),

or exposure to fluorodeoxyuridins (9), cytosine arabinoside (9), dibutyryl cAMP (15) or x-irradiation (33) that are known to retard the growth rate and promote morphological transition, induce higher levels of acetylcholinesterase .

Although acetylcholinesterase levels increase in stationary phase

cells, the induction of acetylcholinesterase can be uncoupled from neurite formation (35) ; furthermore, large increases in activity are induced during logarithmic growth by acetylcholine (36) .

This induction is dependent on

de novo protein and RNA synthesis (9,34) .

Choline acetyltransferase activity

also shows an increase in stationary phase culture (32) .

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TABLE I Biochemical

Inducing

Activity

Agent

Stimulation

Growth Rate

Ref

Acetyl cholinesterase

-serum

25x

decreased

33,9

ACh

37x

unaffected

35

Bt 2cAMP

22x

decreased

15

Bt 2cAMP

4x

decreased

36

5x

decreased

36

Bt 2 cAMP

31x

decreased

37,38

papaverin

65x

decreased

38

Choline acetyltransferase

x-ray Tyrosine hydroxylase

Dopamine 0hydroirylase COMT

none

39

-serum

0

decreased

33

-serum

4x

decreased

44

-serum

-2x

decreased

44

glutamate dehydrogenase lactate dehydrogenase

The activity of tyrosine hydroxylase, the first enzyme in the biosynthetic pathway for production of catecholamines, was shown to remain constant when adrenergic clones of peuroblastoma were induced to differentiate by the addition of cytosine arabinoside, reduction of serum, or x-irradiation (9,17) .

However, a 30-65 fold increase in the enzyme is seen when the cells

are treated with either dibutyryl cAMP, phosphodiesterase inhibitors or sodium butyrate (38,39) . found in some clones (40) .

In addition, the enzyme dopamine-ß-hydroxylase is The storage of catecholamines in neuroblastoma

cells is suggested from histochemical fluorescence (41), the presence of dense core vesicles in electron micrographs (2) and the incorporation of radioactive tyrosine into dopamine and to a lesser extent norepinephrine

Vol . 16, No . 11 (2,42) .

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Neuroblastoma

Additional studies have shown the incorporation of radioactive

tryptophan into serotonin (42,43) . The shift from neuroblast to neuron morphology also results in increased oxygen consumption and 70% and 380% increases in malate dehydrogenase and glutamate dehydrogenase activities respectively (44) .

Not all enzymes show

increased activity in stationary phase neuroblastoma cells, the catechol-omethyl transferase activity remains constant (34) while thymidylate synthetase (45) and lactate dehydrogenase (44) activities decrease . Neuroblastoma cells also have been used in hybridization studies by fusing them, with the use of Sendai virus, to L cells .

With this fusion

it is possible to explore the expression of the differentiated state . Although most differentiated functions are extinguished in hybrid cells, the neuroblastoma x L cell hybrid was found to be an exception.

These

hybrid cells after 10-40 generations possess electrically excitable membranes as well as high levels of acetylcholinesterase and are able to extend neurites that are Bodian stained (46,47) .

These results show that

rapidly dividing cells still retain their ability to express their differentiated functions .

Moreover, hybrids were found that were defective

in some of these neuronal properties .

Cross-correlation of neuronal proper-

ties retained by the hybrids suggested a possible sequence of neuron maturation (47) .

Due to the abnormal karyotype of the mouse neuroblastoma

clones (90-190 chromosomes) there may have been extensive reorganization of genetic units on the chromosomes, thus limiting the usefulness of neuroblastoma .

However, somatic cell hybrids provide a powerful tool and if

applied to normal neuron x established cell line hybrids may reveal the genetic base to neural organization . While some regard neuroblastoma as an abnormal neuron that has added limited knowledge to our understanding of neurobiology, clonal cells of neuroblastoma have helped bring the technology of molecular and cellular biology to the neurosciences .

Although achievements thus far have not

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Vol. 16, No . 11

unlocked the secrets of neural organization many more questions will certainly be asked of these cells and other cell cultures .

Probably, the most important

contribution of neuroblastoma thus far is the new interest, excitement and blood it has brought to neurobiology .

However to quote one astute observer

"It's sure not like E . coli!"

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