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Cell Regulation and Signalling Group, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom
ABSTRACT
To determine the role of the FHIT tumor suppressor gene product, a diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase, in the regulation of the concentration of Ap3A and diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in vivo, the levels of the adenosine(5')triphospho(5')nucleoside (Ap3N) and adenosine(5')tetraphospho(5')nucleoside (Ap4N) families were measured by luminometry in a number of human cell lines and correlated with the expression of Fhit determined by immunoblotting. Fhit-positive cells had no Ap3N or a very low level of Ap3N, whereas most Fhit-negative cells had Ap3N in the range 0.2–0.9 pmol/106 cells. Ap4N (mean value, 0.17 pmol/106 cells) did not correlate with Fhit expression. The results suggest that Fhit efficiently metabolizes Ap3A and Ap3N but not Ap4A or Ap4N in vivo.
Introduction
Abnormalities in the expression or structure of the human FHIT gene, which spans the FRA3B fragile site at 3p14.2, have been observed in a wide variety of common carcinomas and may represent one of the earliest changes in neoplastic transformation (1–3) . Such tumors and derived tumor cell lines express little or no Fhit protein. Reexpression of Fhit protein by transfection or transduction of such cells greatly reduces their tumorigenicity in nude mice, suggesting that Fhit has a tumor suppressor function (4, 5) . In vitro, Fhit has Ap3A3 hydrolase activity (6) . Curiously, this activity does not appear to be required for tumor suppression because a mutant Fhit that binds but does not hydrolyze Ap3A is as effective as the wild type (4, 7) . This has led to the proposal that a Fhit-nucleotide complex may be a component of a new antiproliferative signaling pathway (7, 8) . Nevertheless, because Fhit binds and hydrolyzes both Ap3A and Ap4A (a nucleotide commonly deemed to be associated with proliferation) in vitro (6) , any model of Fhit function should take this into account. The very few measurements of intracellular Ap3A concentration that have been made in eukaryotes (9) suggest it to be present at a level 5–10-fold higher than that of Ap4A; however, no correlations with Fhit expression have been made. Here, we report measurements of Ap3N and Ap4N made in a range of Fhit-positive and Fhit-negative cell lines and show that, in the majority of cases, Ap3N (and thus Ap3A) is undetectable in Fhit-positive cells but is readily assayed in Fhit-negative cells, whereas the level of Ap4N (and thus Ap4A) is not influenced by Fhit expression. The significance of this with regard to Fhit-mediated signaling is discussed.
Materials and Methods
Human Cell Lines.
The U2020 small cell lung carcinoma and RC8 renal carcinoma cells were
generously provided by Dr. K. Huebner (Kimmel Cancer Center,
Philadelphia, PA). HRT18 rectal adenocarcinoma, RT112 bladder transit
cell carcinoma, 2780 ovarian carcinoma, and I407 embryonic jejunal
epithelial cells were from the Oncology Research Unit, University of
Liverpool (Liverpool, United Kingdom). Calu3 non-small cell lung
carcinoma cells were the gift of Dr. C. Walker (Clatterbridge Cancer
Research Trust, Wirral, United Kingdom). Adenovirus 5-transformed 293
kidney cells were purchased from European Collection of Animal Cell
Cultures (United Kingdom), and HL60 promyelocytic leukemia cells were
provided by Dr. D. M. Tidd (Department of Biochemistry, University of
Liverpool, United Kingdom). All cell lines were grown in
recommended media containing 10% FCS.
Nucleotide Assays.
The assay used detects mixed nucleotides of the form
Ap3N and Ap4N, where N is
any base, and not just the diadenosine nucleotides. For each
determination, six 90-mm dishes of cells were grown to about 80%
confluence. Cells were counted in two dishes, and cells in the
remaining four dishes were extracted separately as described
below. Cell layers were washed briefly with 4 ml of warm
serum-free medium, the medium was removed rapidly, and 3 ml of ice-cold
0.4 M trichloroacetic acid were added. Cells were
scraped into a cold tube, the dishes were rinsed with two 1-ml portions
of trichloroacetic acid, and the combined 5-ml extract was left
at 4°C for 15 min. Five ml of 0.6 M
tri-n-octylamine in 1,1,2-trichlorotrifluoroethane were
added, and the tube was shaken for 5 min and then centrifuged at
1,000 x g for 5 min. The upper aqueous layer
(4.4 ml) was mixed with 110 µl of 2 M Tris-HCl
(pH 8.5), 0.2 M magnesium acetate, and 10 units
of shrimp alkaline phosphatase (Boehringer, Germany) and incubated for
60 min at 37°C to hydrolyze mononucleotides. Next, 100 µl of a 50%
(v/v) DEAE-Sephacel suspension in 20 mM Tris-HCl
(pH 7.6) were added to adsorb the remaining nucleotides. After a 10-min
shaking, the suspension was centrifuged for 1 min at 10,000 x g. After discarding the supernatant, the pellet was
washed with three 1.5-ml portions of water and then shaken for 5 min
with 0.5 ml of 1.0 M triethylammonium bicarbonate
(pH 7.5) to elute the dinucleotides. After centrifugation for 1 min,
the supernatant was removed, the pellet was reextracted in the same
manner, and the combined supernatants were freeze-dried. After
redissolving in 400 µl of 25 mM HEPES-NaOH (pH
7.8), 5 mM magnesium acetate and 3 units of
shrimp alkaline phosphatase were added, and the tubes were incubated
for 30 min at 37°C, followed by a 15-min incubation at 65°C.
Each sample was then split in two, and the Ap3N
and Ap4N content was measured in each by
luminometry as described previously (10)
. In cases where
no Ap3N was detected by the above-mentioned
procedure, results were confirmed by repeating the extraction with a
greater number of cells (six 140-mm dishes) and using appropriately
adjusted reagent volumes. For calculations, the recovery of nucleotides
from cell extracts was assumed to be 100%.
Western Blot Analysis.
Analysis of Fhit protein expression was carried out by Western
blotting. Cell lysates were obtained essentially as described
previously (6)
. Samples of lysate containing 15 µg of
protein were then subjected to SDS-PAGE in a 15% gel. The gel was
blotted onto nitrocellulose, and the membrane was blocked overnight at
4°C in PBS containing 0.03% (v/v) Tween 20 and 3% (w/v) dried milk
(blocking buffer). Fhit protein expression was detected with a
polyclonal rabbit antibody raised against a synthetic COOH-terminal
peptide of Fhit (ZP54; Zymed, South San Francisco, CA). The blot
was incubated in primary antibody at a concentration of 0.25 µg/ml
for 3 h at room temperature, washed with blocking buffer, and then
incubated for 1 h in a 1:5000 dilution of peroxidase-conjugated
goat antirabbit IgG (Bio-Rad). The blot was then washed in PBS
containing 0.03% (v/v) Tween 20, and the Fhit protein was
visualized using the enhanced chemiluminescence detection reagent
(Amersham, Little Chalfont, United Kingdom).
Results and Discussion
The level of Fhit protein expression was determined in a number of
tumor cell lines and in nontumor 293 kidney cells by Western blotting
using an antibody raised against a COOH-terminal Fhit peptide. This
shows Fhit as a Mr 16,800
polypeptide. Because inactivating point mutations have rarely been
described in the FHIT gene, the presence of the full-length
Fhit protein is taken to indicate an active Ap3A
hydrolase. This analysis revealed levels of expression varying from
strong to moderate (293, U2020, RC8, HRT18, I407, and Calu3 cells) to
low or undetectable (HL60, RT112, and 2780 cells; Fig. 1
). High expression of Fhit protein in 293, U2020, and Calu3 cells is in
agreement with previous data (11, 12)
.
|
). These values are similar to previous data, which typically measure
Ap4A or Ap4N in unstressed
cultured cells within the range of 0.1–0.5
pmol/106 cells with extreme values of 0.05 and
7.5 pmol/106 cells (9)
. The
differences are largely a function of cell volume; for example, U2020
cells are very small, whereas RC8 cells are large. Given a typical cell
volume of 2 pl, 1 pmol/106 cells equates roughly
to a concentration of 0.5 µM. There was no indication
from our results that the level of Ap4N was
affected in any way by the degree of Fhit expression. In contrast, the
level of Ap3N was markedly affected by Fhit
status in the majority of the cells. Fhit-negative cells (HL60, RT112,
and 2780 cells) had readily detectable Ap3N
(0.2–0.9 pmol/106 cells) at a level above that
of Ap4N (Table 1)
; however, in most Fhit-positive
cells, Ap3N was either undetectable (293, U2020,
and RC8 cells) or very low (HRT18 cells), indicating that Fhit is an
effective Ap3N hydrolase in vivo and
that it normally maintains a very low level of intracellular
Ap3A and Ap3N.
|
; Table 1
). The most probable explanation is that before passage 29,
these cells were Fhit positive (no Ap3N) with one
normal and one defective FHIT allele and that during
culture, loss of the second allele occurred spontaneously in one cell.
The growth advantage conferred by this loss allowed a more rapid clonal
expansion of the homozygous deletant so that it gradually replaced the
original cells during subculture. Thus, the proportion of Fhit-negative
(high Ap3N) cells increased with passage number.
Unfortunately, this could not be tested directly because the culture
was lost at passage 37, and the finding could not be repeated. Although
it is unusual to report an irreproducible result, such a lack of
reproducibility would not be surprising if the underlying cause was a
random mutational event. It is highly improbable that the trend of
increasing Ap3N seen in Fig. 2
could be due to
experimental error; therefore, these data are included. Fhit-positive
I407 cells also had a high level of Ap3N (Table 1)
. In this case, no change in the
Ap3N:Ap4N ratio was noted
with increased passage number. These cells may have been particularly
responsive to a serum factor in the growth medium that stimulated
Ap3N synthesis. IFN is one such factor that is
known to increase intracellular Ap3A
(13)
.
|
How do these results impact on proposed models for Fhit-mediated signaling? Reexpression of Fhit in Fhit-negative tumor cells leads to an accumulation of cells in S phase and an increase in apoptotic cells (5, 17) , whereas an increase in intracellular Ap4A may also induce apoptosis (18, 19) . Brenner et al. (8) have recently proposed that the ground state of Fhit is a Fhit-PPi complex and that an increase in the level of diadenosine 5',5'''-P1,Pn-polyphosphate displaces PPi, generating a Fhit-diadenosine 5',5'''-P1,Pn-polyphosphate complex that signals cell death. Our results would suggest that this complex is most likely to be a Fhit-Ap4A complex. Although increased Ap4A is associated with proliferating cells, there is no direct evidence that it stimulates or is otherwise required for proliferation per se as is often assumed; indeed, one of us (A. G. M.; Ref. 20 ) has recently proposed its involvement in S-phase checkpoint control, regulating replicon initiation and apoptosis after DNA damage in a manner reminiscent of p53. Ap4A is better suited as a positive effector of Fhit function than is Ap3A due to the 80-fold lower catalytic constant of Fhit for the former substrate and the consequent longer lifetime of the proposed Fhit-Ap4A signaling complex (6) . Thus, an increase in Ap4A above the normal level would promote the still poorly defined Fhit-mediated responses, which may include proliferation, checkpoint control, or apoptosis, whereas an increase in the more rapidly degraded Ap3A would counter these responses not by positive signaling but by occupation of Fhit in a futile turnover reaction, thus favoring cessation of proliferation and differentiation (13, 18) .
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by a grant from the North West Cancer
Research Fund. 
2 To whom requests for reprints should be
addressed, at Cell Regulation and Signalling Group, School of
Biological Sciences, University of Liverpool, Life Sciences Building,
Liverpool L69 7ZB, United Kingdom.
3 The abbreviations used are: Ap3A,
diadenosine
5',5'''-P1,P3-triphosphate;
Ap4A, diadenosine
5',5'''-P1,P4-tetraphosphate;
Ap3N, adenosine(5')triphospho(5')nucleoside;
Ap4N, adenosine(5')tetraphospho(5')nucleoside.
Received 12/28/99. Accepted 3/16/00.
REFERENCES
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