Cancer Letters, 4 (19~8} 147--153 147

Elsevier/North-Holland Scientific Publishers Ltd.

REGULATION OF AN ASPARTYL-tRNA SPECIES IN

BHK CELLS IN CULTURE AND IN SOLID TUMOR FORM.
II. THE E F F E C T OF D I F F E R E N T SERA ON ASPARTYL-tRNA IN CULTURE

M.V. MARSHALL, D.L. ADAMS and A.C. GRIFFIN

Department o f Biochemistry, The University of Texas System Cancer Center, M.D. Anderson
Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, Texas 77030 (U.S.A.)
(Received 5 December 1977)
(Accepted 7 December 1977)

SUMMARY

In normal tissues aspartyl t R N A is present in 3 separable components by RPC-

5-chromatography, 2 small and early eluting peaks and a major c o m p o n e n t desig-
nated rRNA~I~P. A late eluting peak, t R N A ~ p has also been found in m a n y
tumors of h u m a n and animals origin, and in BHK 21/13 cells grown in culture,
where 27% or more of the total aspartate acceptance occurs in this peak. In
contrast, normal tissue studied, i.e., hamster liver or kidney, exhibits less than
2.0% o f the total aspartyl tRNA as the late eluting tRNAIA~p. Increasing the
level of fetal calf serum in the medium from 5 to 15% resulted in a change from
0.79 to 1.83 in the ratio of tRNAIAI~P--tRNAIA~ p in BHK cells. Utilizing sera
from different species, the percentage of tRNAIAx~p was found to vary between
26.8 and 66.0% while the combined percentages of tRNAA~ p and tRNAIA~p
varied between 59.5 and 80.8%. It appears that the increased tRNAA~ p was
derived from tRNAIA~ p and vice versa indicating that there may be a close
structural relationship between tRNAA~ p and tRNAIA~p. It was also observed
that BHK t u m o r cells or ascitic fluid enhanced the appearance of tRNAIA~p
in normal host liver.

INTRODUCTION

Changes in the chromatographic profiles have been observed in one or more

o f the aminoacyl tRNAs in all of the tumors that have been extensively studied
thus far [ 1,4]. The significance o f these altered tRNAs, however, in terms of
the origin o f or the function of t u m o r cells has not been established. One of

Abbreviation: BHK, baby hamster kidney.

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the isoaccepting species that may be important in this respect is a late eluting
aspartyl tRNA reported concurrently by Briscoe et al. [3] and Gailagher et al.
[4] to be present in several tumors of animal and human origin. Subsequently,
it was observed that the relative amounts of tRNAA~ p, the major component
of most cellular systems, and tRNAIA~p in baby hamster kidney (BHK) cells
could be regulated by the cellular environment [2]. The further observation
that the relative amounts of tRNAA~ p Iv comprised a constant percentage of
the total BHK aspartyl tRNA led to the hypothesis by Briscoe etal. [1] that
these 2 isoaccepting species are related in that one may be an enzymatic modi-
fication of the other. This communication reports the effect of the serum
source and concentration upon the relative amounts of tRNAA~ p and tRNAIA~p
in BHK 21/13 cells grown in tissue culture.

MATERIALS AND METHODS

BHK 21/13 cells [17] were generously supplied by Dr. Ralph B. Arlinghaus,
Biology Department, The University of Texas System Cancer Center, M.D.
Anderson Hospital and Tumor Institute. Sera were purchased from Grand
Island Biological Company, Grand Island, New York. BHK 21/13 cells were
grown according to Briscoe et al. [2] with the various sera as indicated. At
confluency, each roller bottle contained approx. 5 108 cells. Purification
of total cellular tRNA Was performed according to Briscoe et al. [3]. Pre-
paration of the aminoacyl-tRSlA synthetase enzyme from hamster liver,
which was used in these studies, has been described by Goldman et al. [5] ;
aminoacylation with [ 14C] - or [3H] aspartic acid was accomplished as describ-
ed in Briscoe et al. [3]. RPC-5 chromatography was performed as described
by Briscoe et al. [3]. [3H]- and [14C] aspartic acid, 12,000 mCi/mmol and
208 mCi/mmol, respectively, were purchased from Schwartz/Mann, Orange-
burg, New York.

RESULTS

The relatively large amount of the late eluting aspartyl tRNA (tRNAIA~p) in
the BHK cells grown in culture is shown in Table 1 wherein this component
may approach 40--66% of the total aspartyl acceptance activity. This may be
contrasted to the negligible level of this component observed in normal tissues
(i.e., 1.5 and 0.5% for normal hamster liver and kidney respectively, Table 3).
Increasing the level of fetal calf serum in the culture medium from 5 to 15%
did result in a decrease from 43.4 to 27.4% in the levels of tRNA Asp I v " This
decrease was compensated by a corresponding increase in tRNAA~ p while the
initial smaller peaks of aspartate acceptance remained relatively unchanged.
When newborn calf or calf sera were substituted for the i0% fetal calf serum
there was an even greater proportion of the tRNAIAx~p, Table 1. Dialysis of the
fetal calf serum did not greatly alter the quantity of the late eluting component
but there was a doubling of tRNA~I sp in the cells grown in this serum.
 149

TABLE 1

E F F E C T S OF C A L F SERUM ON ISOACCEPTING ASPARTYL-tRNA P R O F I L E OF

BHK 21/13 CELLS

Percent of total aspartyl-tRNA

acceptor activity

Peak number

Sera I II III IV III/IV

5% Fetal calf serum 8.5 13.9 34.2 43.4 0.79

10% Fetal calf serum 8.7 11.2 40.6 39.6 1.03
15% Fetal calf serum 11.0 11.4 50.2 27.4 1.83

10% Newborn calf serum 4.8 15.9 25.5 53.8 0.47

10% Calf serum 6.6 11.0 16.4 66.0 0.25
10% Dialyzed fetal calf serum 9.3 22.9 32.6 35.2 0.93

a Dialyzed 24 h vs. 60 vol. 0.9% NaC1 then filter sterilized.

Table 2 presents a compilation o f the results obtained when the BHK cells
were grown in a variety o f different sera. While a relatively high percentage of
the late eluting t R N A ~ p is a constant feature, the various sera did produce
some shifts or changes in all of the isoaccepting aspartyl tRNAs. Of interest is
the almost complete disappearance of t R N A A [ p (9.9%) when the cells were
grown in culture containing human serum. This is the t R N A that predominates
in all of the normal tissues we have studied thus far. While the major emphasis
o f this study was concerned with the relative amounts of tRNA~I~P and

TABLE 2

E F F E C T S OF VARIOUS S E R A ON ISOACCEPTING ASPARTYL-tRNA P R O F I L E OF

BHK 21/13 CELLS

Additional sera tested at 10% Percent of total aspartyl-tRNA

acceptor activity

I II III IV III/IV

Hamster 26.3 9.6 23.2 40.9 0.57

Human 22.9 8.2 9.9 59.0 0.17
Horse 6.1 13.1 16.7 64.1 0.26
Rat 21.3 10.2 21.5 47.0 0.46
Guinea pig 31.5 9:0 32.7 26.8 1.22
Lamb 19.8 8.9 23.5 47.8 0.49
Goat 21.1 9.6 29.7 39.6 0.75
Rabbit 19.3 15.3 26.4 39.0 0.68
Pig 24.6 10.4 22.8 42.2 0.54
150

TABLE 3

E F F E C T S O F BHK 21]13 C E L L S ON H A M S T E R I S O A C C E P T I N G A S P A R T Y L - t R N A
PROFILE

Percent o f total a s p a r t y l - t R N A
a c c e p t o r activity

Peak N u m b e r

I II III IV III/IV
Control
H a m s t e r liver 13.5 2.0 83.0 1.5 55.3
H a m s t e r kidney 14.0 0.5 85.0 0.5 170.0

Tissues-BHK t u m o r bearing animals

Tumor a 16.7 3.9 72.9 6.5 11.2
Liver b 23.6 2.7 68.3 5.4 12.6
BHK-actites liver c 12.6 16.5 62.6 8.3 7.5

a 1 0 ' cellslhamster, t u m o r s isolated at 2 weeks.

b Liver f r o m h a m s t e r injected w i t h B H K cells.
c Ascites fluid was p o o l e d f r o m 2 hamsters with BHK-tumors. The filtrate was centri-
fuged 10 rain at 15,000 X g. T h e supernatant was filtered through a 0.45 a m filter
and injected i n t o 4 hamsters. Hamsters were sacrificed 6 weeks later and livers r e m o v e d
for t R N A isolation.

cpm 3H-Asp
XIO-5
RPC-5 Chromatography
16 3H-Asp-t RNA (BHK-tumor)
5H-Asp-IRNA (BHK/IO% Human Serum) .....
14 "n'T
Molarity
12 NaCI

I0 /Z" 1.0

8 t 0.8

0.6

0.4
4 To.l- b ii
2 AAv,,~ 1 ~f: 0.2

0
0 20 40 60 80 I O0
Fraction
Fig. 1. C o m p a r i s o n o f a s p a r t y l - t R N A profiles f r o m BHK 21113 cells grown in 10% h u m a n
serum or as a solid t u m o r . The f R N A fractions f r o m BHK 21113 cells grown in 10% h u m a n
serum or f r o m a solid t u m o r following s u b c u t a n e o u s injection into hamsters were amino-
acylated w i t h [ 3H] aspartic acid and c h r o m a t o g r a p h e d separately on RPC-5 c o l u m n s accord
ing to Briscoe et al. [ 3 ] .
 151

tRNAA~ p, it is o f interest to note that the BHK cells grown in culture media
with different sera did vary considerably in the amounts of the early eluting
isoaccepting aspartyl tRNAs, especially peak I, which may appear as 2 peaks
in the RPC-5 system [2,10].
A n o t h e r interesting result was the apparent host effect o f BHK t u m o r cells
or ascites fluid in the t u m o r bearing hamsters upon the liver aspartyl-tRNA
profiles (Table 3). Ascites fluid, 20 ml, was pooled from 2 hamsters and centri-
fuged to remove any cells present. An aliquot of the supernatant, 10 ml, was
filtered through a 0.45 micron filter prior to i.p. injection of 2.5 ml into each
o f 4 hamsters. The BHK solid tumors contained 6.5% tRNAIA~p in contrast
to the 50% or higher observed when these cells are grown in culture. The livers
of the t u m o r bearing animals exhibited a level of 5.4% in peak IV and the livers
o f hamsters injected with the ascitic fluid obtained from hamsters bearing BHK
solid t u m o r contained 8.3% of this peak. The level of this same isoaccepting
t R N A is almost negligible in the livers of all normal hamsters previously studied.
Fig. 1 shows a comparison of the relative amounts of the 4 aspartyl t R N A
isoacceptors found in BHK cells from 2 extremes, BHK cells grown in solid
t u m o r form and BHK cells grown in tissue culture with 10% h u m a n serum. This
figure is a composite o f 2 experiments where the [3H] Asp-tRNA from both
sorces was cochromatographed with [14C] Asp-tRNA from BHK cells grown in
10% fetal calf serum.

DISCUSSION

Altered isoaccepting t R N A profiles have been observed in all t u m o r cells

studied, and different profiles also occur in tissues and cells exposed to viral
and chemical carcinogens, tRNAs reportedly have some function in embryo-
genesis, differentiation, cell division, control of m R N A translation, N-terminal
additions o f certain amino acids and cell wall protein synthesis [7,11,13,14,15,
16,18,20]. Many of these reported t R N A associations are based upon the
appearance of isoaccepting tRNAs for specific amino acids during the above
physiological and/or pathological events. Whether the altered isoaccepting
tRNA components are prerequisite to or a consequence of these events is
largely unknown.
The findings o f the current study are three-fold. (1) The existence of a late
eluting aspartyl t R N A in neoplastic cells was verified. Almost 40% of the total
isoaccepting aspartyl t R N A occurred in this form in the BHK 21/13 cells grown
under normal culture conditions (10% fetal calf serum). Very little of this
t R N A has been observed in normal cells studied thus far, (2) The isoaccepting
aspartyl t R N A profile m a y be altered or controlled to a considerable extent by
the cellular environment, as evidenced by the variation due to serum source or
content. (3). The presence of t u m o r cells may produce changes in the isoaccepting
t R N A profiles in the normal tissues of the host animal. While the type or con-
centration of sera used in the BHK culture system does exert a considerable
influence upon the tRNAIA~p content, o t h e r u n k n o w n factors are also involved
152

since even in the most favorable conditions for suppression of tRNAIA~ p forma-
tion, a relatively high c o n t e n t of this species is present, i.e., 26.8% with guinea
pig serum and 27.4% with 15% fetal calf serum.
Aspartyl-tRNA profiles, and more specifically the late-eluting tRNAA~ p has
been studied b y this laboratory [1--3] and others [4,19] as a possible char-
acteristic of cancer cells. One t h e o r y to account for the fairly constant percent-
age of tRNAA~ p an d tRNAIA~ p to the total aspartyl-tRNA may be that one
isoaccepting species is an over- or under-modified form o f the other with the
difference perhaps being as subtle as a single base modification [ 4 , 1 0 ] . Aspar-
tyl t R N A has been shown to contain the modified guanosine base, Q, in the
anticodon in E. coli [6]. In addition, mammalian t R N A contains a modified
Q base, Q* whose structure is n o w determined [8,9].
Katze has p r o p o s e d that the difference b e t w e e n tRNA~I~P and tRNAIA~ p
is that tRNA~I~P contains q and tRNAIA~ p contains G, presumably at the same
position in the anticodon [ 1 0 ] . Studies with rat liver and rabbit liver aspartyl
t R N A demonstrate the presence of Q and Q* which is probably in peak III
since it is the major c o m p o n e n t [ 9 , 1 5 ] . We have previously reported that BHK
tRNAIA~ p contains Q which is in contrast to Katze's observations with SVT2
cells [ 1 2 ] . Studies with Con A-Sepharose indicate that BHK t R N A IAsp_.
contains Q* or Q (data n o t shown). In order to better understand the relation-
ship b e t w e e n these 2 aspartyl tRNAs, structural analyses are in progress in our
laboratory. In conclusion, we have shown that BHK t R N A in culture contains
a high percentage of tRNAIA~ p. The content of BHK tRNAIA~ p is variable
depending on the species of serum used to culture the cells. The percentage
o f t R N A ~ p, although lower in solid t u m o r s resulting from subcutaneous
injection of BHK cells, is still greater than that of normal tissue as is the con:
tent of hamster liver tRNAIA~ p in neoplastic cells and in certain tissue culture
cells is u n k n o w n . Sequential analysis of b o t h tRNAA~ p and tRNAA~ p may
shed some light on their relationship and the role of tRNAIA~ p in neoplasia.

ACKNOWLEDGEMENTS

This study was made possible b y a grant from The R o b e r t A. Welch Founda-
tion (G-035) and b y funds provided b y T.K. Dixon. One of the authors, A.C.G.,
is an American Cancer Society Professor.

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