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1991 78: 2261-2268
Establishment and erythroid differentiation of a cytokine-dependent
human leukemic cell line F-36: a parental line requiring granulocytemacrophage colony-stimulating factor or interleukin-3, and a subline
requiring erythropoietin
S Chiba, F Takaku, T Tange, K Shibuya, C Misawa, K Sasaki, K Miyagawa, Y Yazaki and H Hirai
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Establishment and Erythroid Differentiation of a Cytokine-Dependent
Human Leukemic Cell Line F-36: A Parental Line Requiring
Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-3, and
a Subline Requiring Erythropoietin
By Shigeru Chiba, Fumimaro Takaku, Tsuyoshi Tange, Kyoichi Shibuya, Chie Misawa,
Kiyoshi Miyagawa, Yoshio Yazaki, and Hisamaru Hirai
KO Sasaki,
We have established a new nonlymphoid leukemic cell line
from a patient with myelodysplastic syndrome (MDS), which
progressed t o overt leukemia. The parental cell line and a
subline derived from this line have absolute dependency on
several cytokines for their long-term survival and growth.
The parental line designated F-36P requires granulocytemacrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for continuous growth, while a subline designated
F-36E can be maintained in the presence of erythropoietin
(Epo) alone. When these cytokines are depleted, both the
parental and the subline cells die within several days, even in
medium supplemented with fetal calf serum (FCS). F-36E,
maintained in the presence of Epo, constitutively synthesizes
hemoglobin at a significant level. F-36P, which is usually
maintained in the presence of GM-CSF or IL-3, can be
induced t o synthesize hemoglobin when GM-CSF or IL-3 is
substituted by Epo. The surface marker profile shows that
the F-36P cells are positive for the leukocyte common antigen
(CD45) and some common multilineage markers such as
CD13, CD33. and CD34, and negative for T- and 6-cell
antigens and mature myelomonocytic antigens. However,
some monoclonal antibodies recognizing erythroid and platelet glycoproteins react with these cells. Thus, this cell line has
a multilineage phenotype, suggesting that the transformation event occurred in a multipotent stem cell. It is also
evident that the F-36 cells can be induced t o differentiate into
the erythroid lineage in the presence of Epo. This, t o our
knowledge, is the first description of a human leukemic cell
line that can be stimulated t o synthesize hemoglobin by Epo.
0 1991b y The American Society of Hematology.
E
subtype of myelodysplastic syndrome (MDS).” On October 12,
1989, approximately 5 months after the initial diagnosis, the
disease progressed to overt leukemia, at which time the cells used
to establish the line were obtained from the patient’s pleural fluid.
At the intermediate stage of RAEB in transformation (RAEB-T),
the bone marrow aspirate morphology showed some characteristics
of erythroleukemia (-50% of the nucleated cells were erythroblasts, many of which were multinucleated or abnormally large in
size), but did not meet the French-American-British (FAB) criteria for M6 subtype of acute myeloid leukemia. The cells were
cultivated in flat-bottom 24-well plates (Becton Dickinson Labware, Lincoln Park, NJ) in a-modified Dulbecco’s medium supplemented with 20% FCS with or without G-CSF, GM-CSF, or IL-3.
Morphology and cytochemistry. Light microscopy examination
was performed on Wright-Giemsa-stained cytospin preparations.
Cytochemical staining of myeloperoxidase (MPO), double esterase
by a-naphthyl acetate and a-naphthyl butyrate, and Fe and
periodic acid-Schiff (PAS) staining were performed by a standard
protocol.
Electron microscopy. For the standard procedure, the pellet
was fixed in a 2.5% glutaraldehyde, sliced, and postfixed in 1%
osmium tetroxide. Specimens were dehydrated in ethanol, embedded in Epon, and stained with uranyl acetate and lead citrate. The
STABLISHMENT of several cytokine-dependent human leukemic cell lines has been described in recent
years.’.’ Growth of these cell lines is usually enhanced by
various hematopoietic growth factors and other cytokines.
However, quite rare are the human cell lines that will die
out in a few days in the presence of a sufficient amount of
fetal calf serum (FCS),’ but in the absence of exogenously
supplied cytokines. A previously reported erythroleukemia
cell line, TF-1,’ is one example of such a truly cytokinedependent human cell line, and has proven to be an
important tool for the study of hematopoiesis. Here we
describe the cell line F-36, which has many morphologic,
cytochemical, and biologic features in common with TF-1,
including strict dependency on cytokines, but also has some
distinctive biological characteristics and karyotype. In addition, successful establishment of a subline F-36E makes this
cell line novel. F-36E can grow continuously in the presence
of erythropoietin (Epo), but will die within several days
after the depletion of Epo. Thus, this subline is expected to
be useful in studies of the growth signal transduction
mechanism through Epo, as well as through granulocytemacrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), when used in combination with the parental line F-36P, which can be maintained by GM-CSF or
IL-3, but not by Epo. Moreover, both F-36 cell lines are
characterized by the inducibility of hemoglobin synthesis by
Epo. Therefore, the F-36 cell lines can be used not only to
analyze growth, but also to analyze hemoglobin synthesis
induced by Epo.
MATERIALS AND METHODS
Cytokines. Recombinant human Epo6 and granulocyte colonystimulating factor (G-CSF)’ were a generous gift from KirinAmgen (Thousand Oaks, CA). Recombinant human GM-CSF8was
kindly provided by Schering Plough (Osaka, Japan). Recombinant
human IL-39was supplied by Kirin Brewery (Tokyo, Japan).
Establishment of the cell line. We observed a male patient
diagnosed with refractory anemia with excess blasts (RAEB), a
Blood, Vol78, No 9 (November 1). 1991: pp 2261-2268
From the Third Department of Internal Medicine, and the Department of Pathology, Faculty of Medicine, University of Tokyo, Tokyo,
Japan.
Submitted May 3,1991; accepted July 3, 1991.
Supported in part by a grant-in-aidfrom the Ministry of Education,
Science and Culture of Japan, and a research grant for intractable
diseasesfrom the Minishy of Health and Welfare of Japan.
Address reprint requests to Shigem Chiba, MD, Third Depamnent of
Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191 I 7809-0040$3.00/0
2261
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2262
CHIBA ET AL
platelet peroxidase (PPO) was performed by the method of
Breton-Gorious et al.”
Cytogenetic studies. Three months before obtaining established
cells, 18 mitotic figures of freshly aspirated bone marrow cells were
analyzed by the trypsin-Giemsa G-banding method.” Sixteen
mitotic figures of the cultured cells were analyzed by the same
method 3 months after the culture was started.
Surface marker study. Surface markers were detected by an
immunofluorescence assay using commercial monoclonal antibodies listed in Table 1.
Assay for cell proliferation. Short-term cell proliferation was
examined by a colorimetric assay according to Mosmann.” Cells
were incubated at a density of 1 x 104cells1100 pL in 96-well plates
for various periods in RPMI 1640 medium supplemented with 10%
FCS in the presence or absence of 10 nglmL GM-CSF or IL-3, or
20 U/mL of Epo at 37°C. Next, 3-(4,5-dimethylthiazo1-2-y1)-2,5diphenyltetrazolium bromide (MlT) was added at a final concentration to 0.5 mg/mL. Following a 4-hour incubation at 37T, the
insoluble product was dissolved in isopropylalcohol containing
0.04N HCI. The optical density (OD) was measured at 630 nm. For
dose-response plots, the assay was performed in a similar manner
as described above, except that 2 x lo4 cells were initially plated
and various concentrations of GM-CSF, IL-3, or Epo were added,
respectively. M l T was added after 48 to 72 hours of incubation.
Assay of hemoglobin synthesis. Hemoglobin synthesis was detected by benzidine staining. Time course of hemoglobin synthesis
was plotted as the ratio of peak absorbances of the cytosolic protein
at 414 and 280 nm. The cytosolic protein was obtained as follows.
The cell pellet was washed three times with phosphate-buffered
saline (PBS) without calcium and magnesium salts [PBS (-)I to
remove the reddish color of the culture medium, and was resuspended in distilled water. Cells were lysed by five freeze-thaw
cycles, centrifuged at 15,OOOg, and a clear supernatant was transferred. Delta-aminolevulinic acid (6-ALA) was added at 0.5 to 100
mmol/L to F-36 cells under various culture conditions.
Affinity labeling of GM-CSF receptor on the cells. M n i t y labeling of GM-CSF receptor was performed using a homobifunctional
chemical cross-linker, disuccinimidyl suberate (DSS). The F-36P
cells were transferred to the medium and FCS alone 8 to 12 hours
before the experiment. These pretreated F-36P cells, F-36E cells
(1 x 10’) or U-937 cells (5 x lo6),were incubated with ’Tlabeled
GM-CSF at 2 to 4 nmol/L. Binding reactions, cross-linking
reactions, and analyses by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) were performed as described
previously.14
Table 1. Surface Marker Profile of F-36P Cells
CD
4
5
a
10
13
14
19
25
33
34
41a
42b
45
-
MoAb
% Positivity
Leu-3a
Leu-I
Leu-2a
J5
MY7
MY4
Leu-12
IL-2R
MY9
HPCA-1
Gp2b3a
Gpl b
HLe-1
Glycophoirin A
2.5
0.4
0.3
2.5
99.7-99.9
0.7
0.4
0.8
92.3-97.4
25.3-60.5
53.9-96.9
0.2
95.0-97.0
25.3
Abbreviation: MoAb, monoclonal antibody.
RESULTS
Establishment of F-36, a parental line F-36P and a subline
F-36E. Cells cultured in the presence of GM-CSF or IL-3
gradually started active proliferation, following a lag time of
approximately 1 month, when most of the cells cultured in
the absence of these cytokines had stopped proliferation.
During the subsequent 2 months, the proliferative rate of
the active cells increased, but the cells cultured in the
presence of IL-3 alone stopped proliferation at the end of
the third month and died thereafter. At this time, therefore,
the still actively proliferating cells were all maintained in
the presence of GM-CSF. We found that these cells would
die within several days when transferred to the medium
without any exogenously added cytokines, even if supplemented with FCS, but that they would continue to proliferate in a similar manner when GM-CSF was substituted by
IL-3. This parental line was designated F-36P. When
GM-CSF was substituted by Epo, the cells survived for 1to
2 weeks without active proliferation, followed by the death
of most of the cells. However, after a lag time of an
additional 1 month, dividing cells were apparent in one of
these Epo-substituted flasks. These cells started active
proliferation in the presence of Epo, but would die with the
depletion of Epo. This subline was designated F-36E.
The established parental and subline of the F-36 cells
have been maintained in RPMI 1640 medium supplemented with 10% FCS and GM-CSF or IL-3 (for F-36P), or
Epo (for F-36E), respectively. They had been cultured
continuously for 18 months at the time of submission of this
report.
Morphology and cytochemistry. F-36P proliferates as
adherent cells and as single cells in suspension. F-36E has
much less tendency to adhere to the flask. Both of the F-36
cell lines have an irregular, often nonspheric shape, with
spurs or blebs in suspension. Morphology of F-36P and
F-36E resembled that of bizzare nonlymphoid leukemia
blast (Fig 1A) or immature erythroblast (Fig lB), respectively. Cytoplasmic blebs were outstanding in all cytospin
preparations. Dark nucleoli were often present. Peripheral
splittings of cytoplasmic fragments were observed, some of
which contained amorphous eosinophilic zones continuing
from a perinuclear region (Fig 1A). Mitotic figures were
observed at a frequency of up to 3% to 4%. Multinucleated
cells were present at up to 1%.
Cytochemical stains of MPO, or a-naphthyl and double
esterase, were negative. Fe was negative in the established
cell line, although it was positive on some occasions in some
of the patient’s freshly aspirated bone marrow cells. Some
of the established cells, as well as the patient’s aspirated
bone marrow cells, showed large granular staining with
PAS (Fig IC).
Electron microscopic studies showed that the F-36P cells
exhibited a small number of microvilli, ruffles, and elongated cytoplasmic processes of the cell surface. The cells
showed a round but occasionally multilobulated nucleus
without marginal chromatin and with a few large nucleoli,
and a prominent cytoplasm that contained a moderate
number of rough endoplasmic reticulum, mitochondria,
and occasionally scattered open vesicles, and Golgi appara-
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2263
GM-CSF/IL-3- AND EPO-DEPENDENT SUBLINES
tus of varying sizes (Fig 1D). The cells did not exhibit MPO.
However, only a small population (1% to 2%) of cells
exhibited apparent PPO (data not shown), which may
correspond to the positivity of IIbIIIa antigen as described
below, and thus may suggest some megakaryocytic properties of the cells.
Karyotype. Chromosomal study of the freshly obtained
bone marrow cells indicated the presence of normal and
abnormal clones. Thirteen of 18 mitotic figures showed
normal karyotype (43,XY). Four showed essentially the
same karyotype: -5, -7, -9, -17, -18, 19p+, and some
markers, although some additional random loss was observed in each mitotic figure. The majority of the established cells had a homogeneous karyotype (Fig 2);
43,Y (Xp+, -5, -7, -13, -16, -17, -19, -21,2q-, 9p+,
1Oq+, +4mar).
Surface marker profiles. The surface marker profile of
F-36P is summarized in Table 1. The results were essentially the same in F-36E subline. The cells were positive for
the leukocyte common antigen (CD45) and some common
multilineage markers such as CD13, CD33, and CD34, and
negative for T- and B-cell antigens and mature myelomonocytic antigens. However, monoclonal antibodies recognizing glycophorin A (erythrocyte glycoprotein) or platelet
glycoprotein IIbIIIa reacted with the cells.
Cytokine requirements. The survival and growth of the
F-36 cells are shown in Fig 3. Neither the parental or the
subline cells proliferated in the absence of any cytokines,
and both types of cells started to die within 36 hours despite
sufficient supplementation with FCS. IL-5, IL-6, and G-CSF
had no effect on the survival or growth of the cells, either
alone or in combination with IL-3 or GM-CSF (data not
shown). Both the parental and the subline cells proliferated
dramatically with the addition of GM-CSF or IL-3 at 10
ng/mL.
In F-36P, Epo (20 U/mL) prolonged the survival of the
cells for up to 2 weeks, but these Epo-added cells died
thereafter unless either GM-CSF or IL-3 was added. In
contrast, in the F-36E subline, the proliferation of the cells
could be sustained by Epo, as well as by GM-CSF or IL-3.
Furthermore, observing F-36E cells for a longer period
showed that Epo was the most potent cytokine among the
three, because some populations of F-36E did die in the
presence of GM-CSF or IL-3 alone (data not shown).
Dependence of concentrations of GM-CSF, IL-3, or Epo is
shown in Fig 4. Cytokine requirements for the growth of the
parental line and the subline correlated well with the
survival time course shown in Fig 3. Specifically, the
maximal stimulation by Epo was approximately half that by
GM-CSF or IL-3 for F-36P, but maximal stimulation by any
of the three cytokines was almost equal to the F-36E
subline in short-term assays. A colony formation study
showed almost the same tendency as a characterization by
the colorimetric assay; F-36P formed colonies only in the
presence of GM-CSF or IL-3, but not Epo alone, and F-36E
formed colonies in the presence of any of these three
cytokines, although it did not in the absence of any of these
(data not shown).
Epo-induced hemoglobin synthesis. The F-36E cells
(maintained by Epo) constitutively synthesized hemoglobin
at a significant level by estimation with benzidine staining
(data not shown), by peak absorbance of cytosolic protein
at 414 nm (Fig 5B), and by the reddish color of the cell
pellet (data not shown). The level of hemoglobin synthesis
by F-36P cells (maintained by GM-CSF or IL-3) was
undetectable grossly (as judged from color of the cell pellet)
or colorimetrically (Fig 5A). However, after GM-CSF or
I G 3 was removed and replaced by Epo, F-36P started to
synthesize hemoglobin as shown by the estimation with
benzidine staining, by the reddish color of the cell pellet
(data not shown), and by colorimetry at 414 nm (Fig 5A).
The reverse experiment, the replacement of Epo by GMCSF in F-36E subline cells, seemed to decrease cellular
hemoglobin (Fig 5B), although a significant change was not
observed. &ALA also induced hemoglobin synthesis in
F-36P in the presence of GM-CSF or IL-3 (Fig 5A), and
increased hemoglobin synthesis in F-36E maintained in
Epo (Fig 5B). However, F-36P was not induced to synthesize hemoglobin by sufficient concentrations of Epo when
GM-CSF or IL-3 coexisted even at a low concentrations
(0.01 ng/mL).
GM-CSF receptor on F-36 cells. GM-CSF receptor was
identified by affinity labeling using '"I-GM-CSF. The result
was similar to our previous experiments using other leukemic cell lines: two major bands composed of the a-chain
and the P-chain, respectively, were labeled.'4"6 However,
the ratio of the intensities of the two bands were found to
be different from the cells we studied previou~ly.'~~'~
As
shown in Fig 6, in the U-937 cells, the intensity of the lower
molecular weight band originating from the a-chain was
approximately 10-fold greater than the higher molecular
weight band originating from the P-chain (lane a) when a
high concentration (2 to 4 nmol/L) of '%GM-CSF was
incubated, as described by us previou~ly.'~~'~
In contrast, the
lower molecular weight band identified in either the parental or the subline of F-36 was much fainter in comparison to
the U-937 cells (lanes e and c). Comparison of the GM-CSF
receptors on F-36P and F-36E by the ligand affinity labeling
showed no significant difference between these two sublines.
DISCUSSION
A small number of cytokine-dependent or cytokinerequiring human leukemic cell lines have been reported
However, with some exceptions, most of them
can be maintained even in the absence of any exogenous
cytokines if enough FCS is supplemented, although various
hematopoietic growth factors and other cytokines enhance
their growth. It is apparent that the human hematopoietic
cells are much more difficult to establish as cytokinedependent cell lines compared with the murine cells, which
are easily established with dependence on various cytokines, especially IL-3. This difference has been attributed to
frequent intrinsic viral integration in mouse cells or may
represent the different physiologic roles of cytokines between mouse and human. The previously reported erythroleukemia cell line TF-1 is one example of such a truly
cytokine-dependent human leukemic cell line.
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2264
CHIBA ET AL
Fig 1. Morphology of F-36 cells (A through D).
Wright-Giemsa stains of (A) F-36P and (B) F-36E. (C)
PAS staining of F-36P. and (D) a transmission electron microscopic photograph of F-36P (original magnification x9.800; bar, 1 pm).
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2265
GM-CSFIIL-3- AND €PO-DEPENDENT SUBLINES
Y
\\
a
f3gl. (Cont'd).
D
As judged from Wright-Giemsa and PAS stains, the F-36
cells seem to have characteristics of immature erythroid
cclls. Although the host paticnt from whom F-36 was
dcrivcd was initially diagnoscd with MDS by FAB classification, his discasc had many aspects of crythrolcukemia as
dcscribcd in Materials and Methods. The morphological
/
similarity with immature erythroid cclls was not unexpcctcd.
F-36 has complcx chromosomal abnormalities. Among
thc karyotypic ahnormalitics of F-36, -5 and -7 arc both wcll
documcntcd as an abnormality in MDS." The deletion of
chromosomc 17 has hccn infrequently dcscribcd in MDS,
W2.
K a ~ o f F - 3 6 c f ~ l h . A m a j o r k a r y o t y p . w n 4 3 , Y ( X p-5,
+ , -7, -13, -16, -17, -19, -21,2q-,9p+,lOq+,
+4mar).
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2266
CHIBA ET AL
0.8
FQBP (factor -)
* F-36P(GM-CSF)
-
0.6
P
0
---t
0.4
F-36P(IL-3)
F-36P(Epo)
F56E (factor -)
F-36E (OM-CSF)
--*--*-
0.2
0.0
F-36E (11-3)
F-36E(Epo)
0
2
4
6
8
10
Days
but may be of interest since the malfunction of the p53
antioncogene located on chromosome 17q has recently
been demonstrated in a number of tumors, including some
leukemias." The difference of karyotype between the primary cells and the cell line could be explained as further
divergence, such as additional chromosomal loss (2q-,
-13, -16, -19, and -21) or duplication of chromosomes
(9p+ and lo¶+).
F-36 was found to have some features of a megakaryocytic lineage. Platelet glycoprotein IIbIIIa was detected and
PPO was positive, although at a low level. However, the
platelet glycoprotein Ib was negative, and we did not obtain
any other evidence that the cells are megakaryocytic or
Fig 3. Survival end proliferation of F-36P and
F-36E in the presence or absence of hematopoietic
growth factors. The results shown are a mean of
triplicate data. Tho experiments were performed3,6,
and 14 months after the primary culture with similar
results.
differentiate into more mature megakaryocytic cells. Further studies are necessary for detailed characterization of
the lineage and differentiating potency of these cells.
Successful establishment pf F-36E, a subline of F-36P,
makes the F-36 cell line unique. We did not find significant
biochemical differences between the two lines; GM-CSF
receptors identified by affinity labeling were indistinguishable in F-36P and F-36E. However, F-36E differs from
F-36P in that it can grow continuously in the presence of
Epo alone. Thus, F-36E must have some mechanism that
allows it to proliferate in the presence of Epo alone, an
issue that can be clarified by future studies. This may be due
to an alteration of Epo receptor, as was recently reported,"
F-36P
P
0
0.4
A
1
.2
0
V."
.1
1
10
2
20
200
GM, IL-3 (nglml)
EPO (Ulml)
F-36E
,
I
P
0
Fig 4. Responsivenass of F-36 sublines to hematopoieticgrowth factors. The results are shown as
a mean of triplicate data.
B.
OM, 11-3 (nglml)
0
.2
2
20
200
EPO (u/ml)
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GM-CSFIIL-3- AND €PO-DEPENDENT SUBLINES
-
2267
F36P (maintained with 11-3 before day 0)
5-
8
4--
X
a
b
-w
-
.
c d. - .e . f
Epo+8AU
-EPo
1L3+6AU
N
a
3
2
1
0
A
4
Days
F36E (maintained with EPO before day 0)
-
+
-
*
0
u-937
F-36E
F-36P
X
.
-. *I
Y
B
Fig 6. U g m d .tRnity-labellng of the GM-CSF receptors on F-36
cells. The specific bands were obwrved at 150 Kd and 90 to 100 Kd in
at1 three cells liner, F-36P (lane e), F-36E (c), and U-397 (a), respectively. However, the relative intensities of these two bands were
similar in F-36P and F-36E. but different in U-937.
'
.
l
I
0
1
I
I
2
3
Days
1
I
4
5
FIg 5. Hemoglobin s m h by F-36 celh. Time c o u m of the ratio
of the peak absorbance at 414 nm and the absorbance ut 280 nm for
cytosolic protein was plotted. Similar results were obtained by
benzidine staining.
or to convcrgcncc of a cellular mechanism downstrcam
from thc Epo rcccptor and thc rcccpton for GM-CSF or
IL-3 in F-36E. In any caw, thc cstablishmcnt of an
Epo-dcpcndcnt continuous human ccll linc dcscribcd for
thc first timc herc will hc an important tool for thc study of
growth through Epo and its rcccptor. Thc two F-36 ccll
lincs may also bc uscful in thc analysis of the relationship of
growth signals mediatcd by GM-CSF, IL-3, and Epo.
Only onc murinc ccll linc has previously bcen dcscrihcd
that synthcsizcs hcmoglohin in rcsponsc to Epo alonc. Wc
now rcport thc fint human ccll linc in which hcmoglohin
synthesis is stimulatcd hy Epo alonc. Although thc diffcrcntiation potcntial of Epo has hccn provcn ahsolutcly in
colony assay studics using primary bonc marrow cclls, thcrc
arc only limited cxpcrimcntal systcms that use a continuous
ccll linc to study thc diffcrcntiation potcntial of Epo. Thc
F-36 ccll lincs can providc a uscful systcm for thc study of
diffcrcntiation, as wcll as growth mcdiatcd by Epo. and are
cxpcctcd to bc strong adjuncts for understanding thc cntirc
signal transduction mcchanism mcdiatcd by Epo, Gltl-CSF.
and IL-3.
ACKNOWLEDGMENT
We thank leharu Yamamki for electron microscopic studies. We
also thank Tomoaki Kuwaki in Kirin Brewery for his assistance.
and Arinohu Tojo. Seiji Ogawa. Yutaka Ilanazono. and Iliroaki
Honda for fruitful discussion.
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I. Avanzi GC. Lista P, Giovina7zo €3. Miniero R. Saglio G,
Benetton G. Coda R. Cattoretti G. Pegoraro L: Selective growth
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2. Lange .L Valtieri M. Santoli D. Caracciolo D. Mavilio F,
Gerperlein 1, Griffin C, Emanuel B. Finan J, Nowell P, Rovera G:
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