The Transfer RNA Methylases of Human

From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
The
II.
Transfer
RNA
Delayed
Induction
Patients
With
By
Treatment
of
patients
kemia
with
(CLL)
(PHA)
resulted
Methylases
by
in
H.
AND
RIDDICK
from
a fivefold
leu-
increase
The
cies not present
As with normal
PHA
induction
coincided
of fully
markedly
had
tients
L
in
identical
an
P1 IA
of
lymphocytes.2
The
comparison
an (1 the
lymphocytes
tRNA
inethylase
activity
ml.
Fifty
of
in
Ervthroe-ytes
)
the
Icukocytes
purify
to
the
on
lymphocytes
Cells
were
From
the
National
Section
M.D.:
National
BLooo,
on
H.
Tumor
Head,
VOL.
5,
37,
1970;
diflerential
per
Control
October
with
the
induc-
I)eril)herdl
blood
CLL
in
PITA
the
and
induction
subjects
of
with
of
this
untreated
units/mI.,
report.
at
had
patients
CLL
The
1 hour
for
all
at
175
were
Co.
plasma
Upjohn
37#{176}C.The
X
g.
No
than
greater
Medium
RMPI
5,
Associate,
Cell
Biology,
National
Cancer
Control
Bethesda,
(MARcH),
culture
activity
centrifuged
1640
Tumor
Human
1970;
Section,
Clinical
3
in
reported
normal
patients
Mechanisms,
formerly
No.
LYMPFIOCYTIC
delayed
(1000
dextran
in
\Ta.;
institute,
induction
attempt
95
WaS
per
cent
(Industrial
Biological
Biology
Cell
Branch,
Md.
recised
on Cellular
enzymes
of
to those pre-
count.
ml.
Ilematologf/
M.D.:
Section
Cancer
and
Bethesda,
RI1)1)IcK,
cent
since
Cellular
three
per
further
106
that
qualitative
enzyme
we
in
are
of
heparin
withdrawn
X
CLL
METhODS
ml.
6
1)100(1
to 3
Institute,
Charlotte.s’ville,
Human
peripheral
August
Submitted
was
lymphocytes
suspended
Cancer
Dsvio
with
methylase
similar
methylase
of
AND
each
1
sedimented
niethylases
tRNA
lymphocytes
from
containing
and
subsequent
paper
demonstration
1)100(1
syringe
were
containing
made
peripheral
a
and
suggest
quantitative
blastogenesis
precediiig
MATERIALS
collected
data
CHRONIC
delayed
the
CLL
in
methylase
in normal
These
WITh
of
RNA
similar
induces
lymphocytes.
transfer
were
induced
in the cells
per cent im-
blood
of
ing to the
is delayed.
PATIENTS
.
and
viously
reported
in normal
lymphocytes
but that the sequence
of events
of PHA
interaction
with
CLL
lymphocytes
lead-
of tRNA
FROM
peripheral
levels
changes
in tRNA
CLL lymphocytes
of
stimuthree
pa-
pattern
initial
were
higher
who had 10
lymphocytes.
( CLL ) undergo
( PHA ) In the
transfer
RNA
( tRNA
)l4ytOhemagglutini11
normal
formation
normal
lymphocytes
PHA.
Although
all
LEUKEMIA
1w
the
induction,
activity
PITA cells, which
is
compared
with
that
YMPIIOCYTES
tiOD
induction.
the time
with
transformed
delayed
occurring
lated
with
the
GALLO
values
patient
mature
in
transfer
RNA (tRNA)
methylase
activity.
Evidence
was
obtained
which
suggests
that the increase
included
enzyme
speprior
to
lymphocytes,
C.
ROBERT
absolute
of one
From
Leukemia
methylase
three
Lymphocytes.
Lymphocytes
Lymphocytic
chronic
lymphocytic
with
phytohemagglutinin
in
PHA
Human
Chronic
DAVID
lymphocytes
of
Section
Institute,
Mechanisms,
accepted
October
of
University
on
Cellular
Bethesda,
Human
1.5,
Virginia
Control
Md.
Tumor
1970.
Methcal
ROBERT
Cell
School,
Mechanisms,
Biology
C.
GALLO,
Branch,
Md.
1971
293
).
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294
RIDDICK
1.-Clinical
Table
Information
Patient
(1)
on
P.W.,
Age
Patients
58
With
(2)
(Male)
Age
Lymphocytic
62
Leukemia
(3)
J.G.,
(Male)
9 months,
History
Chronic
H.C.,
2
no therapy
Age
60
(Male)
months,
no
GALLO
AND
6 months,
therapy
no
therapy
Complications
None
None
Herpetic
WBC/cu.mm.
11gb (Gm. PerCent)
Platelets/cu.
mm.
Lymphadenopathy
Hepatornegaly
Splenomegaly
Immunoglobulins
Abnormal
antibodies
Bone marrow
75,000
5.8
21,000
Marked
Slight
Marked
Low IgA, 1gM
None
Lymphocytosis
85,000
12.5
119,000
Slight
None
None
Low 1gM
None
Lymphocytosis
157,000#{176}
11.7
116,000
Moderate
None
Slight
Low IgA, 1gM
skin
Direct
infections
Coomb’s
Lymphocytosis,
cent
large
15 per
immature
lyniphoreticular
10 per
#{176}
cent
young
lymphocytes
on peripheral
cells
smear.
Laboratories)
containing
30 per cent fetal calf serum
(Grand
Island
Biological
6500 units
each of penicillin
and streptomycin.
Cell cultures
and harvests,
preparations
of cell extracts
and undermethylated
tRNA
methylase
assays
were
performed
as previously
described.2
Co.)
and
tRNA,
and
RESULTS
Lymphocytic
Chronic
Clinical
data
summarized
on
CLL
in Table
A.
1400
Leukemia
Rote
/
-
/
of
0-0
Patients
patients
from
1. All three
tRNA
patients
Lymphocytes
CLL
Lymphocytes
PHA
-
had
lymphocytes
were
untreated,
“high
1.-Rate
and
obtained
count”
are
(greater
Methylotion
CLL
stimulated
200
whom
with
for
20
hours
000
800
w
Fig.
600
I-
40C
200
Ui
B.
a-
Extent
of
tRNA
Methylotion
have
cn 800
I-
8
600
described.2
with
tRNA
enzyme,
tRNA
400
200
been
performed
methylated
z
Lri1IIII
25
50
g
75
PROTEIN/ASSAY
00
extent
of
tRNA
methylation
in CLL
lymphocytes.
Extracts
of lymphocytes
from
Patient
2
(H.C.)
incubated
with
and without
PHA
for
120
hours
were
assayed
for tRNA
methylase
activity.
Details
of the
assay
and
(1
4ug.)
extent
and
excess
(60
Rate
E.
4ug.)
assays
excess
assays
were
coli underand
limiting
with
limiting
enzyme.
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TRANSFER
RNA
METHYLASES
OF
HUMAN
LYMPHOCYTES.
295
II
6
a,
a,
0
z
5
Ui
2
I
0
C
4
I
:
-
2
a
I4
0
;
of
by
lymphocytes
tRNA
PHA
and
re-
lationship
to the time
of transformation
and mitosis.
Peripheral leukocytes
from
three
patients
with
CLL
were
incu-
:
0.
6
4
2.-Time
induction
CLL
in
.
C)
s
2
;;
Fig.
methylase
bated
for
various
PHA.
Per
cent
-
times
with
transformation
0
8
0
U)
z
6
4
4
I-
I
and
t;
mined
lures.
2
24
TIME
50,000
than
( P.W.
)
their
IN
Rate
and
of
of
described.2
Patient
experiment
of
Patient
methylase
from
the
tRNA
values
induces
and
normal
Blastic
small
duration.
3
)
(J.G.
immature
and
in
addition
in
,
lymphoid
cells)
1
Patient
lymphocytes
to
cells
in
blood
peripheral
a
his
(10
the
cent
of
of
activity
1
(P.W.)
and
cells
higher
magnitude
those
in
cent
cent
are
illus-
small,
mature
were
typical,
and
specific
the
of control
as
will
3. These
in
of
tRNA
values.
lymphocyte
but,
Patient
ex-
the
transformed
changes
response
hours
PHA
per
per
(J.G.),
of
qualitative
the
400
was
3
120
lymphocytes
500
and
results
in lymphocytes
were
with
cultures
observed
of
for
PHA
PHA-treated
Patient
in
PITA
approximately
were
and
in
rates
The
measured
without
methylation)
and
was
without
from
was
higher
controls.
activity
of
extracts
had
nonstimulated
cultures
in
methylases
were
lymphocytes
the
CLL
be
data
suggest
lymphocyte
is comparable
ex-
discussed,
to
that
tRNA
the
response
with
PHA
is
that
a
lymphocytes.2
Transformation
delayed
year’s
mature
Patient
methylase
per
In
quantitative
methylases,
CLL
with
(extent
Patient
absolute
PHA
8.2
elevations
tracts
one
nucleated
of
incubated
cells.
capacity
Similar
than
had
total
incubated
1. Cells
the
less
morphologically
marrow.
those
tRNA
whereas
of
J.G.
Methylation
than
in which
Fig.
of
transformed
transformed
activity
tRNA
PHA
lymphocytes,
large
of
2 (H.C.)
in
of
1,
Patient
168
lymphocytes,
cent
Patient
2, HG.;
(hours)
had
bone
were
deterof
culassaythe have
lymphocytes).
methylation
an
trated
and
per
total
Extent
Extracts
tents
)CLL
mature
(15
of
144
PHA
( H.C. )
2
blood
marrow
120
WITH
mm.
Patient
of
cent
96
CULTURE
peripheral
bone
72
WBC/cu.
and
predominance
of
48
smears
of the
P.W.;
3,
0
index
from
Details
been
0
per
mitotic
of CLL
Lymphocytes
transformation
occurring
compared
percentage
by
to
of
cells
that
in
derived
in
normal
from
CLL
PHA
lymphocytes
all
three
cultured
Figure
lymphocytes.1
patients
had
2
reveals
transformed
after
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296
RIDDICK
72
hours
until
with PHA
and that maximum
elapsed.
This is in contrast
in culture
168
hours
had
normal
lymphocytes
conditions.2
Time
of
The
PHA
per
( Fig.
increases
( Fig.
)
1
at
.
maximal
was
at
with
likely
at
formation
of
three
and
CLL
Patient
is high
In
Fig.
ported
in
composite
3
in
criteria
not
cells
in
of
tRNA
since
from
patient’s
we
counting
72-hour
PHA
have
to
situation
The
as
transformed
in
lymphocytes
most
previously,
higher
two
blood
shown
The
(Table
that
both
lymphocytes
in
patients.
peripheral
previously
associated
the
cultures.
mentioned
other
a
transformed
were
activity
were
the
hours,
Activity
cells
cells
methylase
As
72
proportional.
for
activities
this
of
contrast
in
strictly
identical.
those
in
cent
but
activity
developed.
elevations
cells,
used
was
in
induction
peripheral
of
blood
methylase
induction
is markedly
20-
were
methylase
cells
tRNA
5
the
than
PHA
normal
that
it
two
at
with
shown,
presence
1)
tRNA
is the
methylase
lymphoblasts.3
the
of
apparent
the
tRNA
explanation,
PHA
is
apparent
per
times
in specific
already
largest
methylase
various
( rate )
was
methylase
developed
induction
lymphoid
activity
the
tRNA
and
increases
had
tRNA
patients
3 (J.G.)
inimature
the
of
culture
Lymphocytes
for
activity
activity
whereas
fully
PHA
induced
likely
methylase
transformed
of
specific
occur
identical
in CLL
indices,
GALLO
did not
transformation
under
cultured
paralleled
although
that
marginally
initial
most
is
mitotic
transformation
hours,
Thus,
pattern
all
from
120
explanation
The
tRNA
lymphocytes,2
excluded
from
of
the
lymphocytes
extents
morphologic
PHA
Activity
methylase
methylase
hours.
normal
CLL
tRNA
with
Methylase
cells,
in
Only
.
culture
tRNA
transformed
Elevation
168
the
CLL
)
2
which
was
of
measured
in tRNA
time
of
of
were
PHA
40 hours’
Induction
cent
activities
in
after
transformation
to maximum
AND
activities
coincides
delayed,
tRNA
with
I
three
the
comparison
Normal
an idealized
IN
CULTURE
of PHA
lymphocyte
composite
in
delayed
I
previously
with
patients
I
an
with
both;
re-
idealized
CLL.
It
however,
transformation.
T
#{149}
Normol
TIME
Fig.
3.-Schematic
and
CLL
lymphocytes.
CLL
data
represent
the
transformation
correlating
50
activity
is compared
in
with
6OE
-
methylase
lymphocytes2
WITH
induction
data
of the
PHA
(hours)
of tRNA
methylases
in normal
have
previously
been
reported.2
three
patients
in Fig. 2.
is
in
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
TRANSFER
As
RNA
illustrated
tRNA
the
METHYLASES
in
niethylase
in
of
Irevious
suggesting
cell
lvnlphocvtes
tue
of
of cell
to
not
that
concept
cells
l)lastiC
is high.
tRNA
in
levels
“in
and
are
activity
evidence
although
state
not
of
directly
differentiation
and
lymphocytes
is low
lymphocytes
present
data
in
in
are
lend
clerepressed
( with
reconciled
methylase
activity
CLL
be
presented
normal
and
induced
similar
data
tRNA
to the
Both
the
niethylases
of
and
were
these
activity,
cycle,”
Thus,
initial
previously
related
se.1
normal
activity
can
have
he
Inay
per
the
lyniphocytes
methylase
division,
both
How
.
We
tRNA
cells
CLL
)
above
neoplasia
nlature
of
higher
PHA-transfornied
and
cycle”
and
noted
297.
II
values
tissues?48
level
not
are
Conversely,
“u.s
the
and
absolute
of normal
3,
normal
to ral)i(lity
tile
LYMPHOCYTES.
demonstrating
in
that
HUMAN
the
Patient
than
related
3,
rej)Orts
tumors
of
Fig.
activities
exception
with
OF
cells.
blastic
cells
further
1)0th
CLL
these
support
to
and
normal
neo-
cycle.”
DIsCuSSIoN
Some
of
P1-IA
in
the
known
nornial
however,
some
induces
lymphocytes2
and
in CLL
lymphocytes
stimulated
protein,
to
lation
of
cytes.2
tRNA
this
proteins
normal
render
for
thereby
itself
defect
observation
in
by
CLL
receptors
fully
lymphocyte
cell
lympho-
treated
types
incapable
of
synthesizing
PHA
cell
of
at
delayed
as yet
this
cell
trans-
abnormal
to some
lymphocyte
new
the
or
The
the
,
defective
for
PHA
way
)
that
specific
transformed
in
PHA-
under
lynlphocytes
for
the
response.
interest
normal
Since
hypothesized
ill
secondary
Of
CLL
normal
the
of
induction
well
CLL
reason
synthesis
delayed
is apparently
that
than
of
the
in
delayed
already
tRNA’s
lymphocytes
lymphocytes.
Kornfeld
or
proteins
formation
to
are
previously
insufficient
CLL
adding
induction
primary
or
incubation
in trallSfOrlllatiOfl.
modify
late-appearing
new
could
we
to
addition
CLL
Tile
norinal
primary
the
However,
required
delayed
necessary
rate,
methylase
P1IA
for
true,
methylases
sonic
cells.
be
of
synthesis
( in
be
hours
delay
tile
lyiiiphocytes.
to l)e delayed.’-”-1#{176} P1-IA
known
45
the
DNA
cannot
of CLL
is
even
after
in
lymphocytes.
methylases
clearly
may
are
CLL
occur
observed
and
with
and
tRNA
of
mRNA’s
If
in
is correlated
to PHA
methylases
40
hours
RNA,
induction
tRNA
72
been
in CLL
between
by
illCltlCtiOn
response
also
changes
methylases
that
events
have
of these
tRNA
I)riOr
delayed
biochemical
lymphocytes
the
tRNA
undiscovered
regard
is
membranes
the
recent
contain
fewer
membranes.11
ACKNOWLEDGMENT
The
authors
wish
mission
to
his patients.
Collier
for
study
their
aid
to
in
thank
Dr.
The
preparation
Ralph
authors
of
Johnson,
also
the
wish
National
to
Cancer
thank
Mrs.
I.
Induction
Institute,
Elaine
Ray
for
and
per-
Mrs.
Olga
manuscript.
REFERENCES
1.
The
llavemaun,
delayed
cytic
leukemia
vitro.
Proc.
K.,
and
Robin,
of
chronic
response
lymphocytes
Soc.
Exp.
to
Biol.
A.
D.:
lympho-
Med.
PHA
in
127:668,
1968.
2.
transfer
cytes:
lymphocytes.
3.
tivity
Riddick,
RNA
D.
H.,
methylases
and
Callo,
of
human
R. C.:
The
lympho-
Riddick,
Correlation
normal
30:2484,
D.
of
with
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neoplastic
in
normal
37:000.
I-I.,
transfer
growth
and
PHA
by
Blood
and
Callo,
RNA
and
methylase
differentiation
tissues.
Cancer
R.
C.:
acin
Res.
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4. Baguley,
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BIDDICK
B.
specificity
C.,
and
of
Staehelin,
M.:
adenine-specific
transfer
RNA
methylase
in normal
and
leukemic
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Europ.
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6:1,
1968.
5. Gantt,
R., and
Evans,
V. J.: Comparison
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RNA
methylase
capacity
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neoplastic
and nonneoplastic
cell
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Cancer
Res. 29:536,
1969.
6. Hancock,
R. L: Utilization
of L-methionine
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for
methylation
of soluble
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by
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liver
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Res.
27:646,
1967.
7. Mittelman,
A.,
Hall, R. H., Yohn,
D. S., and Grace,
J. T., Jr.: The in vitro
soluble
RNA
methylase
activity
of SV4Oinduced
hamster
tumors.
Cancer
Res. 27:
1409,
1967.
AND
GALLO
8. Tsutsui,
E.,
Srinivasan,
P. R.,
and
Borek,
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methylases
in tumors
of
animal
and human
origin.
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56:1003,
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9. Rabinowitz,
Y.,
McCluskey,
I.
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B. A.: DNA
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activity
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and
leukemic
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tohemagglutinin.
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to
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phy-
57:257,
1969.
10. -,
and
Dietz,
A.
A.:
Effect
of
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dehydrogenases
of lumphocytes
from
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lymphatic
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31:166,
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S.: Decreased
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receptor
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in chronic
lymphocytic
leukemia.
Biochim.
Biophys.
Acta
192:542,
1969.
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
1971 37: 293-298
The Transfer RNA Methylases of Human Lymphocytes. II. Delayed
Induction by PHA in Lymphocytes From Patients With Chronic
Lymphocytic Leukemia
DAVID H. RIDDICK and ROBERT C. GALLO
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