Expression of neuronal phenotypes in neuroblastoma cell hybrids

DEVELOPMENTAL

BIOLOGY

39, 226246

Expression

(1974)

of Neuronal

Phenotypes

in Neuroblastoma

Cell Hybrids F. ARTHUR MCMORRIS’ AND F. H. RUDDLE Department of Biology, Yale University, New Haven, Connecticut

06520

Four series of neuroblastoma cell hybrids were isolated, using as parental cells a human and a mouse neuroblastoma, a human fibroblast, and a mouse L cell line. Hybrid clones were analyzed for karyotype, morphology, acetylcholinesterase, and choline acetyltransferase. Acetylcholinesterase activity, which was present in the neuroblastoma parents but absent in the fibroblasts, showed continued expression in all series of hybrids, and neuronal morphology was expressed in three of the four hybrid series. Choline acetyltransferase, which mediates acetylcholine synthesis in cholinergic neurons, was absent from all parents and hybrids except for one hybrid clone, in which expression of the enzyme was activated. These results are compared with the results, reported elsewhere, of assays of the same hybrids for additional neuronal phenotypes. These phenotypes are electrical excitability and acetylcholine sensitivity, presence of the neuron-specific 14-3-2 protein and steroid sulfatase, and glycosphingolipid composition. Among those phenotypes which continue to be expressed, the level of expression is closely correlated with acetylcholinesterase specific activity, and also with chromosome number. These results are discussed in terms of the genetic regulation of cell differentiation. INTRODUCTION

In vitro hybridization of somatic cells of dissimilar differentiated type has been reported to result in extinction of some differentiated phenotypes, continued expression of others, and even activation of tissue-specific traits in various hybrid combinations (review by Davidson, 1974a,b). This report describes hybridization experiments using mouse C-1300 neuroblastoma cells, which express a wide variety of neuronal phenotypes in tissue culture. The study was undertaken to answer the following questions: (1) If a large number of different phenotypes characteristic of neurons are assayed in such hybrids, will they all be extinguished, or will all continue to be expressed? (2) Will there be any coordinate patterns of expression in cases where phenotypes are expressed? (3) Will any differentiated phenotype absent from both parents be expressed in the hybrids? (4) If hybrid series are obtained between one ‘Present address: The Wistar Institute, 36th Street at Spruce, Philadelphia, Pennsylvania 19104.

neuroblastoma and several different fibroblasts, or between one fibroblast and severa1 different neuroblastomas, will the results be comparable? (5) Will there be any relationship between phenotypic expression and karyotype? The present report describes the morphology, karyotype, acetylcholinesterase activity, and choline acetyltransferase activity in 22 hybrid clones derived from four different hybrid combinations. Many of the same hybrid clones reported here have also been assayed for the neuron-specific protein, 14-3-2, and steroid sulfatase (McMorris et al., 1974), electrical excitability and sensitivity to acetylcholine (Peacock et al., 1973), and glycosphingolipid composition (Yogeeswaran et al., 1973). The aggregate of these results is here discussed in terms of possible models of genetic regulation of cell differentiation. Minna et ~1. (1971, 1972) have also investigated the expression of neuronal phenotypes in neuroblastoma cell hybrids, and their results are compared with those reported here.

226 Copyright All rights

0 1974 by Academic Press, Inc. of reproduction in any form reserved

MCMORRIS AND RUDDLE MATERIALS

AND

Neuronal

METHODS

Parental cells. NA, the mouse neuroblastoma cell clone used as a parent in most of the hybridization experiments, is a subclone of Neuro-2a, a clonal line of C-1300 mouse neuroblastoma cells isolated in this laboratory by Dr. Robert J. Klebe (Klebe and Ruddle, 1969; Olmsted et al., 1970). NA was isolated as a presumed variant deficient for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) by selecting for spontaneous mutants of Neuro-2a resistant to 8-azaguanine (Klebe, unpublished). The absence of HGPRT activity in these cells has been confirmed using the assay system of Shin et al. (1971). One to two cells per 10’ plated in HAT medium (Littlefield, 1964) give rise to colonies, indicating probable reversion to HGPRT+. IMR-32 is a human neuroblastoma cell line established from an abdominal neuroblastoma in a 13-month-old boy by W. W. Nichols, J. Lee, and S. Dwight at the Institute for Medical Research, Camden, New Jersey. We have not succeeded in cloning IMR-32. However, cells resembling fibroblasts have not been observed in our cultures, and the cells are karyotypically very homogeneous with three rearranged marker chromosomes (see below), indicating probable clonal origin of the cell line. The neuronal origin of IMR-32 is best substantiated by its expression of high levels of the neuron-specific protein, 14-3-2 (McMorris et al., 1974). LM(TK-) is a clone of mouse L cells, which were originally derived from adult subcutaneous connective tissue; clone LM(TK-) was isolated by Kit et al. (1963) as a nonreverting variant lacking thymidine kinase (TK). MRC-5 is a normal diploid line of human skin fibroblasts derived from an adult male, and was obtained from Dr. Frank Perkins of the Medical Research Council, London. Tissue culture. Tissue culture media,

Phenotypes

in Cell Hybrids

227

sera, and other reagents were purchased from Grand Island Biological Co., Grand Island, New York. NA, MRC-5, and LM(TK-) cells were maintained in Dulbecco-Vogt modified Eagle’s medium supplemented with 10% -y-globulin-free newborn calf serum, 100 U/ml penicillin G, 100 pg/ml streptomycin sulfate, 3 x 10m6M FeSO,, 1 x lo-“ M CuSO,, and 5 x lo-’ M ZnSOr (DV-10). HAT medium was prepared from DV-10 by the addition of lo-’ M hypoxanthine, 4 x lo-‘M aminopterin, and 1.6 x 10m6M thymidine (Littlefield, 1964). Cells grown in DV-10 or HAT were maintained in a humidified atmosphere of 10% CO*, 90% air. IMR-32 cells were maintained in Eagle’s minimal essential medium, supplemented with serum, antibiotics, and minor salts as above, and with 10 mM each of L-alanine, L-asparagine, Laspartic acid, Gglutamic acid, L-proline, and L-serine, and 3.8 mM glycine (MEM-lo-NEAA), and kept in a humidified atmosphere of 5% CO?, 95% air. IMR-32 was found to grow poorly in DV-10 or in HAT prepared from DV-10; this greatly aided selection against parental IMR-32 cells during hybridization even though IMR-32 bears no known enzyme mutations. Sendai virus was grown in the chorioallantoic sac of g-day embryonated chicken eggs (Spafas, Inc., Norwich, Connecticut), harvested and concentrated as described by Okada and Murayama (1968), and inactivated with 0.05% /?-propiolactone (Fellows-Testagar, Detroit, Michigan) by the method of Neff and Enders (1968). Cells were hybridized in monolayer, essentially by the method of Klebe et al. (1970), and hybrids were selected by the HAT system of Littlefield (1964). Hybrid clones were isolated with stainless steel cloning rings and maintained in HAT medium. The full HAT selection system was used to isolate hybrid series NL-I; the other three series were isolated using half-selection (Davidson and Ephrussi, 1965).

228

DEVELOPMENTALBIOLOGY

Chromosome methods. Chromosome preparations were made from Colcemidarrested metaphase cells essentially by the method of Moorhead et al. (1960). Four different staining methods were used: standard 1.5% aceto-orcein; the method of Pardue and Gall (1970) and Chen and Ruddle (1971) for constitutive heterochromatin; and the Giemsa banding method of Sumner et al. (1971) and the quinacrine mustard fluorescence banding method of Caspersson et al. (1971) for maximum resolution of chromosome detail. Well-spread metaphases with good morphology were photographed, and individual chromosomes identified and ideograms constructed. Human chromosome identification followed the 1971 Paris Conference Convention. 2 Cell morphology. Cells were observed in plastic tissue culture vessels and photographed in the living state under phasecontrast optics. In all cases cells were cultured in the presence of 10% serum. Based on the number of neurites per cell, the length and degree of branching of neurites, and the frequency of cells with neurites, each clone was given a numerical score which ranged from 0 for nonneuronal morphology (as in parental fibroblasts) to 4 for very neuronal morphology. (The criteria for assigning scores are listed in Table 1.) A more rigorous assessment of morphology was not attempted because of the sensitivity of morphology to numerous conditions of cell culture and the unreliability of morphology alone as an index of cell differentiation. The morphological scores used here are primarily for purposes of comparison. Preparation of cell extracts. Cells previously maintained in logarithmic growth for 5-7 generations were inoculated into plastic tissue culture dishes (Falcon Plastics Co.). Parameters of feeding and transfer *IVth International Conference on Standardization in Human Cytogenetics, Paris, 1971.

VOLUME39. 1974

were carefully controlled. Cells were harvested 12 days after confluency was reached. Immediately prior to harvest, cells were counted either visually using a ruled grid in the microscope eyepiece, or by dispersing the cells in a duplicate culture with trypsin solution and counting with a Coulter counter. Visual and Coulter counts were in close agreement. Cell monolayers were rinsed three times and collected by scraping into a small volume of cold 50 mM KPO,, pH 6.8. Cells were ruptured by sonication, and frozen in aliquots at - 90°C for subsequent assay. Enzyme activities were unchanged by one freezing and thawing or by frozen storage for up to 7 months. Protein was determined by the method of Lowry et al. (1951). Cell extracts for isozyme analysis were prepared in the same way, except that cells were sonicated in distilled water, and were harvested within a few days of reaching confluency. Vertical starch gel electrophoresis and specific enzyme staining were performed as described elsewhere (Ruddle and Nichols, 1971; Edwards et al., 1971; Nichols et al., 1973). Acetylcholinesteruse assay. Acetylcholinesterase (AChE; EC 3.1.1.7) was assayed by a modification of the method of Wilson et al. (1972). “C-Acetylcholine iodide, 2.43 Ci/mole, was purchased at >98% purity from New England Nuclear, Boston, Massachusetts, and was lyophilized before use. Unlabeled acetylcholine iodide was purchased from Sigma Chemical Co., St. Louis, Missouri; Dowex 50-X8 from Bio-Rad, Richmond, California; Triton-X-100 from Packard Instruments, Downers Grove, Illinois; and standard “‘Ctoluene and fluors from New England Nuclear. BW284C51 was the generous gift of Dr. Howard J. Schaeffer of Burroughs Wellcome, Inc. Research Triangle Park, North Carolina. The assay mixture consisted of 50 mM KPO,, pH 6.8, 0.5% Triton-X-100, 3.3 mM acetylcholine iodide, specific

MCMORRIS

AND RUDDLE

Neuronal

activity 0.428 Ci/mole, 0 or 2 x 10e7 M BW284C51, and O-9 mg/ml homogenate protein, in a total volume of 50 ~1. This concentration of the drug 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51) has been shown to give essentially complete inhibition of “true” acetylcholinesterase (EC 3.1.1.7), but little inhibition of “pseudo” cholinesterase (EC 3.1.1.8) or other esterases hydrolyzing acetylcholine but not specific to neurons (Aust.in and Berry, 1953; Klingman et al., 1968). AChE as reported here is that portion of homogenate-dependent acetylcholine hydrolysis that is sensitive to 2 x 1O-7 M BW284C51. The reaction was carried out for 10 minutes at 37°C. Product “C-acetate was separated from substrate on Dowex 50 ion-exchange resin, Na+ form, and counted by liquid scintillation in Triton X-100 based scintillation fluid (Patterson and Greene, 1965). Quench corrections were made by the channels ratio method, and blank values were substrated. The reaction was linear with respect to time and protein concentration. Choline acetyltransferase assay. Choline acetyltransferase (ChAT; EC 2.3.1.6) was assayed by a modification of the method of Schrier and Schuster (1967) and Wilson et al. (1972). Acetyl-1-‘C coenzyme A, 59.2 Ci/mole, 99% pure, was purchased from New England Nuclear, and lyophilized before use. Choline chloride, neostigmine methyl sulfate and choline&erase (type IV, from horse serum) were obtained from Sigma Chemical Co.; unlabeled acetyl coenzyme A (trilithium, trihydrate, 91% pure) from P-L Biochemicals, Milwaukee, Wisconsin; and Dowex -1-X-8, Cl- form, from Bio-Rad. Sources of other materials have been cited above. The final reaction mixture consisted of 0.22 mM acetyl-1-“‘C coenzyme A, 5.43 Ci/mole, 2.5 mM choline chloride, 0.1 mM neostigmine methyl sulfate, 0.2 M NaCl, 50 mM KPO,, pH 6.8, and O-20 mg/ml

Phenotypes

in Cell Hybrids

229

homogenate protein, in a total volume of 50 ~1. The reaction was carried out for 20 min at 37°C. Products were separated from substrate on Dowex 1, and counted by liquid scintillation as described above. Quench corrections were made and blank values subtracted. The reaction was linear with respect to time and protein concentration. Acetylcholine was identified as the product of the ChAT reaction by high-voltage paper electrophoresis, essentially by the method of Potter and Murphy (1967) as reviewed by Jenden and Campbell (1971). Reaction products were separated at 18 V/cm in 1.5 M acetic acid-O.75 M formic acid buffer, pH 2.0, with authentic 14Cacetylcholine and “C-acetyl coenzyme A as standards. The products were localized with a Vanguard Model 880 radiochromatogram scanner and quantitated by liquid scintillation counting. The percentage of the radioactivity eluting from Dowex 1 that was acetylcholine was computed, and this correction was applied to the results. RESULTS

Isolation of Hybrid Clones, and Confirmation of Hybrid Nature. Four series of hybrid clones were isolated: mouse neuroblastoma NA x mouse L cell LM(TK-) (hybrid series NL-I); NA x human fibroblast MRC-5 (series NM-V and MM-VII); NA x human neuroblastoma IMR-32 (NI-I); and IMR-32 x LM(TK -) (IL-I and IL-II). Well-isolated clones were picked with stainless steel cloning rings and maintained in HAT medium. In most instances, only one clone was isolated from each culture flask, to ensure their origin from independent fusion events. In cases where two clones were isolated from the same flask, they were designated by the same number followed by a different letter (as NL-I-7A and NL-I-7B).

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DEVELOPMENTALBIOLOGY VOLUME~~,1974

The hybrid nature of the clones was confirmed by both karyotype and isozyme analysis. Figure 1 illustrates the electrophoretic pattern of glucose phosphate isomerase (GPI; EC 5.3.1.9) in representative clones of the mouse x mouse NL-I hybrids and their parents. The presence in the hybrid cells of the hybrid heterodimeric form as well as the two parental homodimeric forms of the enzyme (DeLorenzo and Ruddle, 1969) establishes that the GPI genes from both parents are present and active within the same cell. Figure 2 illustrates the electrophoretic pattern of glucase-6-phosphate dehydrogenase (GGPD; EC 1.1.1.49) in the NM hybrids and their parents. Again, hybrids express both parental forms plus an intermediate heteropolymer. The NI-I-2 hybrid clone also expressed a hybrid GGPD pattern. In the fourth series, IL, two clones expressed only the mouse forms of all marker enzymes tested, although the presence of human thymidine kinase was inferred from their ability to grow in HAT medium. Confirmation of their hybrid nature thus rested more heavily on karyotype analysis (below). The

other two IL clones together exhibited both the human and mouse forms of GPI, glutamate oxaloacetate transaminase, lactate dehydrogenase subunits A and B, mannose phosphate isomerase, and peptidase B. In sum, the human x mouse hybrid clones expressed both human and mouse forms of 15 constitutive enzymes, including the above plus adenosine deaminase, indophenol oxidase A, malate dehydrogenase, malic enzyme, nucleoside phosphorylase, peptidase C, phosphoglycerate kinase and phosphoglucomutase-1. Their hybrid nature is thus amply confirmed. Morphology of hybrid cells. Hybrid clones in the NL-I series showed a spectrum of morphology, ranging from nonneuronal cells strongly resembling normal fibroblasts, to cells in which the length, degree of branching, and number of neurites per cell equaled or exceeded the neuroblastoma parent. Parents and representative hybrid clones are shown in Figs. 3-5. Hybrids of the NM and NI series, in which mouse neuroblastoma cells were hybridized with human fibroblasts or human neuroblastoma, respectively, were neu-

FIG. 1. Glucose phosphate isomerase (GPI) isozymes in NL-I hybrids and parents, separated by starch gel electrophoresis. “0” indicate s origin. NA cells contain the A, or slow form of GPI, while LM(TK-) cells contain the allelic B form. Mixtures of NA and LM(TK-) exhibit only the two parental forms of the enzyme. The presence of the hybrid heterodimer band in addition to the parental homodimers in the NL-I hybrids is dependent on the activity of both the A and B aheles within the same cell (DeLorenzo and Ruddle, 1969).

MCMORRISANDRUDDLE

Neuronnl

% FIG. 2. Glucose-6-phosphate dehydrogenase (GGPD) isozymes in NM hybrids and parents, separated by starch gel electrophoresis. MRC-5 exhibits the human form of GGPD, while NA exhibits the mouse form. Hybrids between NA and MRC-5 (NM hybrids) exhibit both parental forms plus the mouse-human heterodimer. This confirms their hybrid nature.

ronal in morphology although less so than the mouse neuroblastoma parent. Hybrids between the IMR-32 human neuroblastoma and mouse LM(TK-) cells were epithelioid or fibroblastoid in morphology; this is consistent with their predominantly murine chromosome constitution (Fig. 5). The morphological appearance of the pa-

Phenotypes

in Cell Hybrids

231

rental and hybrid clones on a numerical scale of 0 to 4 is summarized in Table 1 and in Figs. 3 and 4. Criteria for judging neuronal morphology are given in Table 1. Acetylcholinesteruse activity. Table 2 summarizes the acetylcholinesterase (AChE) activity present in the neuroblastoma cell hybrids and parents. AChE is present at high activity in the mouse neuroblastoma NA, at lower activity in the human neuroblastoma IMR-32, and is very low or absent in LM(TK-) and MRC-5. The neuroblastoma with weaker activity has greater than 25-fold more AChE activity than the strongest fibroblast; hence the difference between the neuroblastoma and nonneuroblastoma parents is clear-cut. In hybrids between cells expressing and cells not expressing AChE, the enzyme continued to be expressed, although in some clones the specific activity was reduced to only 4% of the neuroblastoma parent. Mixtures of NA and LM(TK-) homogenate gave exactly intermediate enzyme activities, indicating that LM(TK-) does not contain an inhibitor of AChE. The specific activity in the hybrids was always at least 300-fold higher than the fibroblast parent and so the continued expression of AChE is unequivocal. AChE also showed continued expression in the neuroblastoma x neuroblastoma hybrid NI-I-2, in which AChE was expressed by both parents. Two exceptions to this continued ex.pression were observed. Clones IL-I-BOA and IL-I-BOB failed to express AChE, and also had lost over 95% of the chromosomes of the human neuroblastoma parent. The absence of AChE is thus possibly the result of loss of genes for AChE. Two other hybrid clones of the same parentage show continued expression of AChE, in spite of the great preponderance of nonneuroblastoma chromosomes. True AChE activity (EC 3.1.1.7) was distinguished from the activity of other esterases present in the homogenates by the AChE inhibitor, BW284C51. In NA,

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FIG. 3. Morphology of NA and LM(TKm) parental cells, grown in the presence of 10% serum and photographed in the living state, unstained, with phase-contrast optics. (A-C) NA mouse neuroblastoma cells; morphological score = 3. (D) LM(TK-) mouse L cells; morphological score = 0. Criteria for assigning morphological scores are described in Table 1.

MCMORRIS AND RUDDLE

Neuronal Phenotypes in Cell Hybrids

233

FIG. 4. Morphology of representative clones of the NL-I hybrid series, grown in the presence of 10% serum and photographed in situ, unstained, by phase contrast. (A) NL-I-1; morphological score = 0. (B) NL-I-11A; score = 1. (C) NL-I-7A; score = 2. (D) NL-I-15B; score = 3. (E) NL-I-20; score = 3. (F) NL-I-17; score = 4. Criteria for assigning morphological scores are described in Table 1.

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VOLUME 39.1974

whether AChE was expressed at a high or low level. The mean for the NL-I hybrids was 90.5%, and for the NM clones, 86.4%. Among the IL hybrids expressing AChE, one clone showed 81.6% true AChE. Choline acetyltransferase actiuity. Choline acetyltransferase activity (ChAT) is summarized in Table 3. The enzyme, which is characteristic of cholinergic neurons only, was not detected at appreciable levels in any of the parental cell lines or in any of the hybrid clones except one, NMVII-lo. In NM-VII-lo, the activity of ChAT is more than loo-fold greater than the baseline activities measured in the parenTABLE I NEURONAL MORPHOLOGY IN NEUROBLASTOMACELL HYBRIDS AND PARENTS Clone

FIG. 5. (A) IMR-32 human neurohlastoma cells, and (B) hybrid clone IL-I-ZOA, derived by fusing IMR-32 with LM(TK-). Unstained preparation; phase contrast.

91.1% of the total choline&erase activity was AChE, while in LM(TK-) and MRC-5, 0% and 31.9% of the almost negligible total cholinesterase was apparently AChE. Thus, presence of AChE as a high percentage of the total cholinesterase can also be considered an index of neuroblastoma differentiation. By this criterion IMR-32, in which AChE represents 58.8% of the total cholinesterase, is not as highly differentiated as NA. In all hybrids expressing AChE except IL-11-16, at least 80% of the cholinesterase was AChE, regardless of

MorpholWY”

NA LM(TK-) (NA x LM(TK-)): NL-I-1 3 5 7A 7B 8 10A 11A 11B 15A 15B 17 18 20

3 0 0 1 3 2 3 4 0 1 0 3 3 4 0 3

Clone

MorpholWY”

MRC-5 0 (NA x MRC-5): NM-V-11 2 VII-7 2 VII-IO 2 IMR-32 2 (IMR-32 x LM(TK-)): IL-I-20A 0 I-20B 0 II-5 0 II-16 0 (NA x IMR-32): NI-I-2 3

D Criteria for assignment of morphological scores: 0 = fibrohlastic or epithelioid; no neuronal cells. LM(TK-) and MRC-5 are type cell lines; 1 = occasional cells with neurites in a predominantly nonneuronal population; 2 = higher incidence of neuronal cells than 1, but neuronal cells not as frequent, or not as neuronal, as 3; 3 = several processes at least one cell diameter long present on 25% or more of the cells under normal logarithmic growth conditions. NA is the type clone; 4 = all neuronal characteristics (length, branching, and number of neurites per cell, and frequency of such cells in a population) consistently exhibited to a higher degree than 3. See Figs. 3-5.

MCMORRIS

AND RUDDLE

Neuron& TABLE

ACETYLCHOLINESTERASE

Clone

NA LM (TK-) (NA x LM(TK-)): NL-I-1 3 5 7A 7B 8 10A 11A 11B 15A 15B 17 18 20 MRC-5 (NA x MRC-5): NM-V-11 VII-7 VII-10 IMR-32 (IMR-32 x LM(TK-)): IL-I-2OA I-20B II-5 II-16 (NA x IMR-32): NI-I-2

ACTIVITY

Phenotypes

235

in Cell Hybrids

2

IN NEUROBLASTOMA

CELL HYBRIDS

AND PARENTS

SD*

Number of measurementsc

% of Total ChE activity

106.61 0 (-0.05)

10.49 0.03

12 9

91.1 0

3.96 8.41 301.31 9.52 174.49 145.76 9.24 4.75 6.01 51.46 230.46 242.10 4.54 66.67 0.15

0.13 0.34 3.96 0.63 14.15 14.93 0.49 0.68 1.11 1.98 7.67 17.26 0.78 2.05 0.02

6 6 6 6 6 6 6 6 6 6 6 9 6 3 6

88.5 92.1 91.1 93.4 88.4 89.4 91.2 89.1 93.5 94.8 89.8 90.1 90.8 84.5 31.9

45.35 54.20 100.03 4.08

3.09 2.55 6.16 1.20

6 5 6 9

85.6 87.8 85.7 58.8

0.12 0.21 0.24 0.20

6 6 6 6

0 0 81.6 46.3

1.14

3

82.7

True AChE, (nmoles/mg/min)”

0 (-0.04) 0 (-0.27) 5.62 2.54 23.70

“Nanomoles of acetate released from acetylcholine per milligram of homogenate protein per minute of incubation at 37°C. True AChE is that portion of total cholinesterase activity that is sensitive to 2 x lo-’ M BW284C51. b Standard deviation. c Two to three cultures were assayed the total number of times indicated.

tal cells. Thus, the genes responsible for the expression of ChAT appear to have become activated in the NM-VII-10 hybrid. Whether these were the human or mouse genes has not been determined. Mixtures of homogenates of NM-VII-10 and NA gave exactly intermediate activity for ChAT, indicating that NA does not contain an inhibitor of ChAT activity, and also that there was no interference due to AChE during the ChAT reaction. Assay of AChE under the conditions of the ChAT

assay (i.e., with the omission of Triton X-100 and the addition of 0.1 mA4 neostigmine methyl sulfate and 20 rM acetyl-l“C iodide, incubated for 20 min at 37°C) also indicated that there was no measurable interference due to AChE. The material identified as “‘C-acetylcholine by electrophoresis of the products from NM-VII-10 was hydrolyzed completely by purified horse serum cholinesterase. Karyotypes of parental and hybrid clones. The numbers and morphology of

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TABLE 3 CHOLINE ACETYLTRANSFERASEACTIVITY IN NEUROBLASTOMACELL HYBRIDS AND PARENTS Clone

ChAT (pmoles/mg/min)”

(W 0fSmDean)”

NA LM(TK-)


19 14

(NA x LM(TK-)): NL-I clones (13 clones)


NL-I-18 MRC-5


5-32 (average: 16) 8 18

6 9


20 14 14 25

6 12 12 6


12-24 (average: 20)

(NA x MRC-5): NM-V-11 VII-7 VII-10 IMR-32 (IMR-32 x LM(TK-)): IL-I and IL-II clones (4 clones)

1.35

Number of measurementsC 15 6 5-9 per clone

6-12 per clone

a Choline acetyltransferase activity is recorded as picomoles of acetylcholine produced per milligram of homogenate protein per minute of incubation at 37”C, as corrected by direct recovery of the acetylcholine product by paper electrophoresis (see Materials and Methods). ’ Standard deviation, as percent of the mean. c Two to three cultures of each clone were assayed the total number of times indicated.

the chromosomes in NA and LM(TK-) cells were determined by counts from photomicrographs of 27-53 well-spread metaphases. The normal karyotype of NA is shown in Fig. 6 and LM(TK-) in Fig. 7. Chromosome numbers of the parents and NL-I hybrids are summarized in Table 4. Unique marker chromosomes could be found in each parent (Figs. 6-8), and markers from both parents could be detected in all clones of hybrids (Figs. 9 and 10). Figure 8 depicts four marker chromosomes with related appearance which were observed in NA neuroblastoma cells. Their related appearance suggests that they are derived by modification of a single chromosome (probably N-2, the most frequent of the series). Two different chromosomes of this series were never seen in the same neuroblastoma cell. There is thus some heterogeneity within clone NA even though it has been serially cloned three times (Klebe, 1970; Klebe, unpublished). The frequency of marker chromosomes of this

series in NL-I hybrid clones closely parallels their frequency in the NA parent (see Table 4). The most striking characteristic of the chromosome numbers presented in Table 4 is the large differences between hybrid clones of the same parentage. Mean chromosome numbers range from 102 in NL-I-11A to 192 in NL-I-8, almost a 2-fold difference. The expected chromosome number in a hybrid cell derived from two modal parental cells is 139. Six hybrid clones had a chromosome number greater than this; the mean numbers ranged from 151 to 192, in all cases greater than the sum of the means plus standard deviations of the parental populations. It is possible that these clones represent fusions between one cell of one parent with two cells (or one 2s cell) of the other parent, followed by partial chromosome loss. Formation of triple hybrids by fusion of three different cells from genetically and karyotypically distinct lines has been described (Ricciuti and Ruddle, 1971), and some degree of chromo-

6

L-6

FIG. 6. Karyotype of NA mouse neuroblastoma, stained for constitutive heterochromatin. Neurohlastoma marker chromosomes N-2 and N-4 are indicated. N-2 is characterized by a small block of heterochromatin which appears to divide the arm into two equal segments, and N-4 has a large block of heterochromatin that extends nearly to the tip of the long arm. Nine biarmed chromosomes; 94 chromosomes total. FIG. 7. Karyotype of LM(TK^) mouse L cell, stained for constitutive heterochromatin. Marker chromosomes L-l-L-7 are identified as described by Chen and Ruddle (1971). The L-5 marker chromosome resembles a chromosome found in NA cells and thus could not be used as a marker in this hybrid combination. Thirteen biarmed chromosomes; 45 chromosomes total. FIG. 8. Mouse neuroblastoma marker chromosomes of the N-O - N-3 series, stained by the aceto-orcein (upper row) or constitutive heterochromatin method (lower row). Chromosomes of this series were observed in different NA cells, and in clones of hybrids derived from NA. Two different markers of this series were never seen in the same NA cell. Chromosomes in this figure are magnified by different amounts to compensate for different degrees of chromosome contraction in the cells from which they were taken. Compare with Figs. 6, 9, and 10. 237

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DEVELOPMENTAL

BIOLOGY

TABLE KARYOTVPES

Clone

Total chromosomes Mean

NA LM(TK-) sum of above NL-I-1 3 5 7A 7B 8 10A 11A 11B 15A 15B 17 18

20

92 47 139 103 114 136 151 164 192 120 102 126 152 165 166 104 134

OF NL-I

HYBRID

VOLUME 39, 1974

4 CLONES AND PARENTS

Biarmed chromosomes

SDb

Mean

SDb

3 3

10 13 23

2 1

5 7 14 10 10 26 4 14 10 13 13 12 11 4

18

1

20 19 23 22 26 20 16 19 22 23 23 25 19

2 5 2 3 4 1 2 3 2 4 3 5 1

Neuroblastoma markers, N-0-N-3” No./cell

Type c

53 27

N-O N-2 N-2 N-3 N-l N-2 N-2 N-2 N-2 N-2 N-2 N-l

30 49 30 41 41 58 26 51 28 29 35 26 34 59

1

0 1 1 1 0 2 1 1 1 1 1 1 1 1

0 1

-

“Neuroblastoma marker chromosomes of the N-O-N-3 series (see Fig. 8). b SD = standard deviation. ‘Chromosomes N-O, N-l, N-2, and N-3 were present in different NA neuroblastoma frequencies: N-O, 2%; N-l, 9%; N-2, 58%; N-3, 11%; none present, 21%.

some loss is almost universally observed in somatic cell hybrids (Weiss and Ephrussi, 1966; Ruddle, 1972). MRC-5 human fibroblasts exhibit the normal diploid karyotype of the human male. By a combination of the staining methods employed, every human chromosome was readily distinguishable from the chromosomes of the mouse cell lines with the exception of the human 13-15 group. Of these, the presence or absence of human chromosome 14 could be inferred by presence or absence of the human form of nucleoside phosphorylase (Ricciuti and Ruddle, 1973). IMR-32 human neuroblastoma cells have a near-diploid karyotype with a modal chromosome number of 50 * 1. The karyotype includes several chromosomes with altered morphology, including two translocation products of chromosome number 1 and a translocated number 7 chromosome. Each of these chromosomes

Number of cells

cells at these

was readily discernible from all other human and mouse chromosomes, and none were observed in any of the hybrid clones. Chromosome numbers of the 8 human x mouse hybrid clones are presented in Table 5. In all instances, the human chromosome number is highly reduced. Clone NMVII-10 has retained 9 different human chromosomes at high frequency and at least 3 more at lower frequency, yet the total chromosome number in this clone (92 & 4) is the same as the mouse parent (92 k 3). Loss of mouse chromosomes from human x mouse hybrids has also been described by Jami et al. (1971) and more recently by Coon and Minna (personal communication). Hybrid clones NM-VII-7 is apparently of 2s mouse x 1s human composition. The three clones for which detailed analysis of human chromosomes was not performed were all found to express human constitutive enzymes.

9 l-2

L-3

N-4

L-l N-4

L-6

L-4

L-7

N-l

FIG. 9. Karyotype of hybrid clone NL-I-8, stained for constitutive heterochromatin. Marker chromosomes from neuroblastoma and LM(TK-) are identified. Twenty-five biarmed chromosomes; 199 chromosomes total. 239

240

DEVELOPMENTAL

BIOLOGY

FIG. 10. Karyotype of hybrid clone NL-I-3, LM(TK-) marker chromosomes are identified.

stained for constitutive heterochromatin. Neuroblastoma Eighteen biarmed chromosomes; 112 chromosomes total. TABLE

CHROMOSOME

Clone designation NM-V-11 NM-VII-7 NM-VII-10 NI-I-2 IL-I-20A IL-I-20B IL-II-5 IL-II-16

Total chromosomes, +SD” 91 * 4 152 + 9 92 * 4 96 48 50 50 51

zt 8 * 2 * 3 * 2 * 2

VOLUME39.1974

COUNTS OF ~HUMAN

Biarmed chromosomes, *SD”

5 x MOUSE

Sum of parents

HYBRID

138 138 138

34 36 30

18 15 17 16 13

142 97 97 97 97

26 27 19 48 19

l

2 2 2 1 1

CLONES

Number of cells counted

11 * 1 17 * 3 16 * 1 + * * zt

and

Human chromosomes presenP ND ND 1,5,6,7, 11, 12, 14,c 19, x ND 8, 10, 17 17,22 7, 12, 17, 19 d

’ Standard deviation. * Human chromosomes present in greater than 40% of the cells scored. Human chromosomes were scored in cells stained either by the quinacrine mustard or Giemsa banding methods (see Materials and Methods). The number of cells for each clone so scored is: NM-W-10,9; IL-I-ZOA, 45; IL-I-BOB, 37; IL-R-5,70; IL-B-16,31. ND = not done. c Chromosome number 14 inferred to be present because of the presence of human nucleoside phosphorylase (Ricciuti and Ruddle, 1973). dAn intact human chromosome number 17 was identified in 2 of 31 cells scored.

McMoruas

AND RUDDLE

Neuronal

DISCUSSION

Patterns of differentiated expression. When a variety of neuronal phenotypes were assayed in neuroblastoma cell hybrids, three different patterns of expression were observed. The majority of the phenotypes continued to be expressed; these include neuronal morphology and acetylcholinesterase activity as reported here, and also action potential generation, acetylcholine sensitivity (Peacock et al., 1973), and the neuron-specific 14-3-2 protein (McMorris et al., 1974). Steroid sulfatase activity was extinguished in the same hybrids (McMorris et al., 1974). Choline acetyltransferase activity was activated in one hybrid clone derived from parents that failed to express this phenotype. Thus most hybrids differed from both parents in the constellations of phenotypes expressed. These results are most consistent with a model in which different phenotypes are regulated by different types of control systems (e.g., positive and negative control). Comparable patterns of phenotypic expression were observed in four different hybrid combinations. Hybrids derived by fusing NA cells with two different fibroblast lines and by fusing LM(TK-) with two different neuroblastomas showed the same patterns of expression, and the quantitative levels of expression in the hybrids all fell within the same range. The results are thus not peculiar to any single hybrid combination. Minna et al. (1971, 1972) have also reported the continued expression of AChE, neuronal morphology, and action potential generation in mouse neuroblastoma x L cell hybrids. The findings discussed here indicate that additional neuronal phenotypes are expressed in such hybrids, that extinction and activation also occur, and that these same patterns are observed when different neuroblastoma and fibroblast parents are used. Coordinate expression. There is a considerable range in the quantitative expression of phenotypes which continue to be ex-

Phenotypes

in Cell Hybrids

241

pressed in the hybrids. AChE activity in the NL-I hybrids ranges from 4% to 280% of the neuroblastoma parent. When the clones are ranked in order of their AChE activity, they fall naturally into two groups, with low and high AChE activity. In general, low or high activity also correlates with low or high activity of the other phenotypes assayed, and also with low or high total chromosome number (Table 6). Clone NL-I-7A shows some characteristics of both the low and high groups. When the activities are plotted against each other, this correlation becomes obvious (Fig. 11). The significance of the correlations was determined by applying Student’s t test to the least-squares correlation coefficient, F, of the parameters compared (Steele and Torrie, 1960) (Fig. 11 and Table 7). In most instances, the correlations are quite good. Neuronal morphology, and percent of cells lacking the L cell hyperpolarization activation response (Peacock et al., 1973), correlate with both log AChE and chromosome number, and log AChE is very closely correlated with chromosome number. 14-3-2 protein (McMorris et al., 1974) is correlated only weakly with chromosome number, but is more closely correlated with log AChE. An exception to this coordinate expression is action potential dV/dt, which is a measure of the strength of action potential generation in the hybrid clones (Peacock et al., 1973). The lack of correlation of dVldt with log AChE is due to two clones, NL-I-7A and NL-I-17, which show low AChE and high dV/dt, and the reverse, respectively. If these two clones are eliminated from the calculations the association between dV/dt and log AChE becomes quite good (0.01 < p < 0.02). Thus the apparent correlation is lacking in these two clones with respect to this phenotype. What this statistical analysis indicates is that in spite of the individual exceptions, such as the “low” clone NL-I-3 that expresses as much 14-3-2 protein as many of the “high” clones, the overall pattern of quantitative expression is nonrandom. In

242

DEVELOPMENTALBIOLOGY

VOLUME 39,1974

TABLE 6 COORDINATE EXPRESSION OF NEURONAL PHENOTVPESIN NL-I Clone

NA LM(TK-) (NA x LM(TKm)): Low NL-I-1 18 11A IlB 3 10A Uncertain 7A High 15A 20 8 7B 15B 17 5

True AChE (nmoles/ mg/min)”

14-3-2 protein (ng/mg soluble protein)”

AP dVldt, (V/sec)C

%HA responsesd

HYBRIDS Neuronal morphol%Y

Chromosome number

107 0

1020 60

30 0

0 97

3 0

92 47

4 5 5 6 8 9

140 0 490 270 530 50

3

92

15 0 6

100 42 46

0 0 1 0 1 0

103 104 102 126 114 120

10

190

59

8

2

151

51 67 146 174 230 242 301

120 220 670 850 580 190 730

22

33

32 20

a 21

8

0

3 3 4 3 3 4 3

152 134 192 164 165 166 136

DClones of NL-I hybrids are arranged in order of increasing AChE activity. b Nanograms of 14-3-2 protein per milligram soluble protein from cultures maintained in confluency for 12 days. Data from McMorris et al. (1974). c Maximum rate of membrane depolarization during action potential generation (action potential dV/dt), volts per second. Data from Peacock et al. (1973). d Percent of cells giving the hyperpolarization activation electrical response, which is characteristic of L cells but not of neuroblastoma cells. Data from Peacock et al. (1973).

contrast with these correlations, hybrid clones differing widely in their expression of neuronal phenotypes show essentially similar glycosphingolipid patterns (Yogeeswaran et al., 1973). The correlation of phenotype with chromosome number may offer some insights. Davidson and co-workers (Davidson, 1972; Davidson and Benda, 1970) and Fougkre et al. (1972) have reported that phenotypes extinguished in 1s x 1s hybrids are expressed when 2s differentiated cells are fused with 1s fibroblasts. NA cells are hypertetraploid, and therefore analogous to the 2s cells used by these workers. In addition, several of the NL-I clones may be derived from fusion of two NA cells with one LM(TK-) cell. The clones listed as

“low” in Table 6 contain 9-26% fewer chromosomes than the number expected, while of those listed as “uncertain” or “high,” two have the expected number (134 and 136; 139 f 6 expected) and the remainder contain more than the expected number of chromosomes. The observed pattern of coordinate expression is consistent with a model in which the neuronal phenotypes are expressed as a block, and their level regulated by a single integrating control system. The correlation with chromosome number suggests that the interclonal differences may be due to segregation of regulatory genes or to some other gene dosage effect on regulatory elements. Individual exceptions to this coordinate pat-

MCMORRISAND

RUDDLE

Neuronal

tern (as low expression of one phenotype in an otherwise “high” clone) suggest that escape from this control can occur, but the statistical significance of the correlations indicates that some factor nevertheless governs quantitative expression of the phenotypes in a coordinate fashion. The possibility cannot be excluded that the interclonal differences are due to heterogeneity within the NA parent, even though NA has been serially cloned three times; however, this explanation is unlikely in view of the close correlation of phenotype with chromosome number, and it would still require a mechanism t.o maintain the coordinate pattern of expression. Another possibility is that in clones with a low level of expression all the activity derives from a few very active cells. This would also necessitate maintenance of coordinate expression. Minna et al. (1971, 1972) have reported the continued expression of acetylcholinesterase, neuronal morphology, and action potential components in hybrids between mouse neuroblastoma and L cells, and have also observed interclonal variation in the level of expression of these phenotypes. When each clone was scored as simply “plus” or “minus” for each phenotype, it was found that the phenotypes could be arranged in a sequence such that a clone expressing a given phenotype also ex-

Phenotypes

243

in Cell Hybrids

pressed all prior phenotypes in the sequence. The authors proposed that this reflects a sequence of steps during neuron maturation and that this indicates that some phenotypes cannot be expressed independently of certain other phenotypes (Minna et al., 1972). Phenotypic expression in the hybrids presented here is more consistent with a

Chromosome

Number

FIG. 11. Coordinate expression of neuronal phenotypes in 14 clones of NL-I hybrids. Acetylcholinesterase is plotted on a semilogarithmic scale vs chromosome number or neuronal morphology, and a straight line fitted to the points by the method of least squares. Criteria for scoring neuronal morphology on a scale from 0 to 4 are described in Table 1. r = least-squares correlation coefficient; t = Student’s t, calculated from r with n-2 degrees of freedom; p = probability, based on the t statistic, that a correlation at least as good as the observed could occur by chance alone.

TABLE 7 COORDINATE

EXPRESSION

OF NEURONAL

PHNEOTYPES IN NL-I HYBRIDS: CHROMOSOME NUMBER

Log AChE activity’ r Log AChE activity 14-3-2 protein Action potential dVldt % HA’ responses Neuronal morphology Chromosome number

0.56 0.17 -0.74 0.91 0.80

P

0.02-0.05 >0.5 0.02-0.05
CORRELATION

WITH ACHE

Amvmy

AND

Chromosome number” r

D

0.80 0.43 0.47 -0.86 0.86 -


’ r = least-squares correlation coefficient; p = probability of obtaining a correlation at least as good as the observed by chance alone, determined by calculating the Student’s t statistic from the correlation coefficient (Steele and Torrie, 1960). ’ HA, hyperpolarization activation.

244

DEVELOPMENTAL

BIOLOGY

scheme of coordinate expression than with a sequential scheme such as proposed by Minna et al. (1972). At the same time, the results of Minna et al. (1972) are largely consistent with the coordinate expression observed here. Further results are needed before it can be determined whether the sequential model of Minna et al. (1972) or the coordinate model presented here is the more realistic. The results reported here do support the observation of Minna et al. (1972) that at least some neuronal traits can be expressed in the absence of others. Actiuation. One neuronal phenotype, choline acetyltransferase, was activated in a hybrid derived from parental clones which both lacked the property. This phenomenon has not been observed previously. The glial-specific protein, S-100, was not activated in any of these hybrids (McMorris et al., 1974). The possibility cannot be ruled out that this hybrid clone arose from a variant NA cell in which ChAT had become activated prior to fusion. Determination of whether the activated genes are mouse or human, or both, may help resolve this question. More recently, activation of ChAT activity has been detected in neuroblastoma x glioma hybrids (B. Hamprecht, T. Amano, and M. Nirenberg, personal communication). Activation of other genes may have occurred in these hybrids. Two clones of IL hybrids express AChE, yet the only human neuroblastoma chromosome present in both of these clones, chromosome number 17, is also present in the two clones that fail to express AChE. These results may be due to retention of the human AChE genes on morphologically altered chromosomes, extinction of AChE in two clones despite gene retention, or activation of the mouse genes for AChE. Identification of the AChE as mouse or human would be instructive. Activation of fibroblast genes for AChE may be responsible for the very high levels of AChE activity in some of the NL-I hybrids.

VOLUME 39.1974

Gene regulation in differentiated cells. Hybridization of neuroblastoma cells with fibroblasts thus results in continued expression of some phenotypes, extinction of others, and in one case activation of a previously unexpressed neuronal phenotype. These results are most easily accommodated by a model in which some tissuespecific phenotypes are under positive control and others are under negative control in differentiated cells. Evidence was also presented for the coordinate expression of neuronal phenotypes. Whether these controls are the same controls responsible for the process of differentiation from an indifferentiated precursor cell, or are more simply responsible for stabilizing the differentiated state once it has been established, cannot be assessed at present. This material has been included by F. A. M. as part of the thesis requirement for the degree of Doctor of Philosophy at Yale University, 1972, and has been presented at the Second Annual Meeting of the Society for Neuroscience, Houston, Texas, October, 1972. The authors wish to thank Ms. Marliss Geissler, Ms. Elizabeth Nichols and Ms. Suzie Chen for their excellent technical assistance, Dr. T. R. Chen and Mr. Richard Creagan for valuable assistance with the chromosome analyses, and Drs. Richard Davidson, Fred Gilbert, and Chester Partridge for critically reviewing the manuscript. This work was supported by N.I.H. Grant 5ROl-GM-09966. REFERENCES L., and BERRY, W. K. (1953). Two selective inhibitors of cholinesterase. &o&em. J. 54, 695-700. CASPERSSON, T., LOMAKKA, G., and ZECH, L. (1971). The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability. Here&as 67, 89-102. CHEN, T. R., and RUDDL~ F. H. (1971). Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes. Chromosoma 34, 51-72. DAVIDSON, R. L. (1972). Regulation of melanin synthesis in mammalian cells as studied by somatic hybridization. IV. Effect of gene dosage on the expression of differentiation. Proc. Nat. Acad. Sci. U.S. 69, 951-955. AUSTIN,

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