2’-deoxycytidine protects normal human bone marrow progenitor
TRANSCRIPT
Blood, Vol 74, No 6 (November 1), 1989: pp 1923-1928 1923
2’-Deoxycytidine Protects Normal Human Bone Marrow Progenitor CellsIn Vitro Against the Cytotoxicity of 3’-Azido-3’-Deoxythymidine
With Preservation of Antiretroviral Activity
By Kapil Bhalla, Martin Birkhofer, Li Gongrong, Steven Grant, William MacLaughlin, John Cole,
Gary Graham, and David J. Volsky
Bone marrow cytotoxicity of 3’-azido-3’-deoxythymidine
(AZT), an anti-human immunodeficiency virus (anti-HIV)
drug, has been attributed to deoxyribonucleotide pool
perturbations that might result in impaired DNA synthesis
in normal bone marrow elements. We examined, in vitro,
the effect of high, but clinically achievable and nontoxic,
concentrations of 2’-deoxycytidine (dCyd) (� 100 gimol/L)
on high-dose AZT mediated growth inhibition and intracel-
lular biochemical perturbations in normal bone marrow
progenitor cells. Colony formation by bone marrow pro-
genitor cells in semisolid medium was significantly pro-
tected by dCyd against the inhibitory effects of co-adminis-
tered, high concentrations of AZT (10 gimol/L). Also, dCyd
significantly corrected AZT mediated depletion of intracel-
lular thymidine triphosphate (dTTP) and dCyd triphosphate
3,-AzIDO-3,-DEOxYTHYMIDINE (AZT) is a 2’,3’-
dideoxynucbeoside analog that is converted intracelbu-
larly to AZT-monophosphate (AZT-MP) by mammalian
thymidine kinase (Km. 3 gzmol/L).”2 AZT-MP is ultimately
phosphorylated to AZT triphosphate (AZT-TP).2 The latter
metabolite is a much better substrate for human immunode-
ficiency virus (HIV) reverse transcriptase (RT) than mam-
malian DNA polymerase.2 AZT-TP competes with thymid-
me triphosphate (dTTP) for incorporation into growing
chain of DNA resulting in chain termination.’ The major
anti-HIV effect is felt to be due to inhibition of RT by
AZT-TP, chain termination of proviral DNA synthesis by
incorporated AZT, or perhaps a combination of the two
effects.’3 At 10 �amol/L bevel, AZT completely protects T
lymphocytes against HIV infectivity.4 In clinical trials, AZT
has been shown to reduce morbidity and mortality and
improve immunologic function in patients with the acquired
immune deficiency syndrome (AIDS).3’5’6 Particularly, in
patients treated with high-dose AZT regimens achieving
peak plasma AZT concentrations between 6 and 10 �imob/L,
cultures of peripheral blood mononuclear cells for HIV
became negative.6 Several studies have demonstrated that at
these and higher concentrations, AZT markedly inhibits the
in vitro growth of bone marrow prognitor cebbs.79 Clinically,
long-term or high-dose administration of AZT results in
serious anemia and leukopenia.6”#{176} Recently, AZT has been
administered as a continuous intravenous (IV) infusion to
children with symptomatic HIV infection, and plasma con-
centrations approximating 5 �imob/L were achieved with a
significant clinical improvement in a majority of patients.”
The major mechanism of bone marrow toxicity of AZT in
humans may be pyrimidine starvation induced by the deple-
tion of intracellular dTTP and deoxycytidine triphosphate
(dCTP) pools in normal bone marrow cells,5”2 although
treatment ofcultured leukemic cells (HL-60 and K-562) and
1-19 cells with high concentrations of AZT (� 10 gimol/L)
has been reported to increase intracellular dCTP bevels.’3
The depletion of dTTP occurs because AZT-MP inhibits the
phosphorylation of thymidine monophosphate (dTMP) (Fig
(dCTP) levels in normal bone marrow mononuclear cells
(BMMC). Moreover. dCyd reduced the intracellular accu-
mulation of AZT triphosphate (AZT-TP) and its DNA incor-
poration in BMMC. In contrast, co-administration of dCyd
(100 �imol/L to 1 mmol/L) did not reverse AZT (10 gimol/L)
mediated suppression of HIV infectivity in HUT-i 02 cells in
culture, although a partial reduction in intracellular AZT-TP
pools and its DNA incorporation as well as a correction of
AZT mediated depletion of dTTP and dCTP pools was
observed in these cells. These studies suggest that dCyd at
high concentrations might ameliorate the bone marrow
cytotoxicity of high-dose AZT without impairing its anti-
HIV effect.
a) 1989 by Grune & Stratton, Inc.
I ),2 The mechanism of dCTP depletion in bone marrow cells
by high concentrations of AZT has not been elucidated, but
appears not to be due to inhibition of ribonucleotide reduc-
tase by AZT-TP.’4
2’-Deoxycytidine (dCyd) is present in human plasma in
concentrations ranging between 0.4 and 2.9 gzmol/L.’5 Intra-
cellubarly, dCyd is phosphorylated by dCyd kinase to dCyd
monophosphate (dCMP).’6 It is ultimately converted to
dCTP, which acts as a substrate for DNA pobymerase.’6
Opposing this phosphorylation, dCMP deaminase converts
dCMP to deoxyuridine monophosphate (dUMP), which is
further metabolized to dTMP by dTMP synthetase (Fig
I )t7.�8 Therefore, exogenously provided dCyd may not only
expand dCTP pools via the salvage pathway, but may also
lead to the expansion of intracellular dTTP pools.18 We have
previously demonstrated that high concentrations of dCyd
(� 100 �zmob/L), which are nontoxic for CFU-GM and are
clinically achievable,’5”9 stimulate and protect normal
human bone marrow progenitor cells against the cytotoxic
effects of dCyd analogues.�#{176}’2’ The aim of the present study
was to examine the effects of high concentrations of dCyd on
the metabolism and cytotoxicity of high-dose AZT in normal
human bone marrow progenitor cells as well as CD4 positive,
From the Division ofOncology, Department ofMedicine. Colum-
bia University College ofPhysicians and Surgeons, New York, NY;
Molecular Virology Laboratory, St. Luke’s-Roosevelt Medical
(‘enter, New York, NY; and Medical College of Virginia, Richmond
VA.
Submitted January 23, 1989; accepted June 28. 1989.
Address reprint requests to Kapil Bhalla. MD. Division of
Hematology/Oncology. Medical University ofSouth Carolina, I 71
Ashley Avenue, Charleston, SC 29425
The publication costs ofthis 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 1 734 solely to
indicate this fact.
© 1 989 by Grune & Stratton, Inc.
0006-4971/89/7406-001 l$3.OO/O
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AZT
I - TdRI (ease
AZT-MP -
I �dTMPG2I- KinaseAZT-DP
TdR d�yd
I �-dTMP dCMP KinasedTMP - dUMP - dCMP
( Synthetase Deaminase ___dCMP
I Kinase
dTDP dCDP
5.ReverseTseas�se��
1924 BHALLA ET AL
AZT-TP dTTP dCTP
� N /� �meuse
Fig 1 . Intracellular metabolism of AZT. thymidine. and deoxy-cytidine. Thymidine (TdR) or AZT are phosphorylated by cellularthymidine kinase to form thymidine or AZT monophosphate
(dTMP or AZT-MP, respectively), which are further phosphory-lated by dTMP kinase to their diphosphate derivatives (dTDP orAZT-DP. respectively). dTDP and AZT-DP are again phosphory-lated by nucleoside diphosphate kinase to form dTTP and AZT-TP.respectively. AZT-TP competes with dTTP as a substrate for DNApolymerase or viral reverse transcriptase for incorporation intogrowing chain of DNA resulting in chain termination. AZT-MPinterferes with its own phosphorylation and with the phosphoryla-
tion of dTMP. Deoxycytidine is phosphorylated by a series ofkinases in succession to form dCyd monophosphate (dCMP).
diphosphate (dCPD). and triphosphate (dCTP). which is also asubstrate for DNA polymerase. Intracellular dCMP is also deami-nated to deoxyuridine monophosphate (dUMP) and further methyl-
ated to dTMP by dTMP synthetase.
cultured T lymphocytes (HUT-102 cells). Since the anabob-
ism of AZT to its phosphorybated derivatives has been
reported to be similar in uninfected and HIV infected cells,2
only uninfected cells were used for these studies. An addi-
tional aim was to determine the effect of dCyd on the
anti-HIV activity of high concentrations of AZT in HIV-
infected HUT-102 cells.
METHODS
Drugs and chemicals. [3HJ AZT (1 2.5 Ci/mmob) and AZT-TPwere kindly provided by Dr Philip Furman of Burroughs Wellcome
Co (Research Triangle Park, NC). dCTP and dTTP were purchasedfrom Sigma Chemicals (St Louis, MO). Drugs were stored as drypowders at - 20#{176}Cand reconstituted in sterile media before use.
Cells and viruses. The origin, cultivation, and characterizationof HUT-102 cells has been described elsewhere.� The suspensioncultures were maintained in RPMI 1640 medium supplemented with5% fetal bovine serum (FBS). N lT isolate of H1V23 was propagatedin CEM cells.24 HUT-102 cells were infected with cell-free, viruscontaining supernatants (mutiplicity of infection being one), as
described previously.25 The viability of HUT-102 cells was deter-mined by trypan blue exclusion method.
Evaluation ofanti-HIV effect by immunofluorescence assay andenzyme immunoassay for p24 antigen. Uninfected or HIV-
infected HUT-l02 cells were incubated with 10 gimol/L of AZTwith or without 100 gimol/L or 1 mmol/L dCyd. At the end of 3 or 7
days of incubation, aliquots of cells were washed in phosphate
buffered saline (PBS), spotted on a slide, dried, and fixed in acetoneat -20#{176}Cfor 15 minutes. The fixed cells were reacted with an HIVreactive, human T-bymphotropic virus (HTLV) type I-non reactiveserum from a hemophilia patient (D-77l) at a 1:20 dilution. This
serum contains antibodies to all of the viral polypeptides, as deter-
mined by Western blot (immunoblot) analysis. After a 30-minute
incubation at 37#{176}C,the cells were reacted with fluorescein isothio-cyanate-conjugated goat anti-human immunoglobin G and readunder an EPI fluorescent microscope (American Optical, Buffalo,
NY).
Antiviral activity was also assayed by the inhibition of theproduction of HIV p24 (gag) antigen in the cell free supernatantmedium of infected cells exposed to different concentrations of
AZT ± dCyd. p24 antigen was assayed using an Enzyme Immu-
noassay (Coulter Immunology, Hialeah, FL) according to the manu-
facturer’s instructions.
Culture ofCFU-GM and CFU-GEMM. Bone marrow aspirateswere obtained with informed consent from normal volunteers. These
studies were sanctioned by the Investigational Review Board ofColumbia University College of Physicians and Surgeons. Mononu-
clear cells were isolated, and adherent bone marrow cells containing90% or greater monocytes and macrophages were removed byincubation for I hour in tissue culture flasks in McCoys 5a mediumwith 10% fetal calf serum (FCS). Nonadherent BMMC were
cultured in semisolid media to obtain CFU-GM and CFU-GEMMcolonies, using previously described techniques.’9�26 The designated
concentrations of AZT with or without the various concentrations ofdCyd were added to the culture medium. After incubation in a fullyhumidified 37#{176}C,5% CO2 incubator for 10 days (CFU-GM), or 14days (CFU-GEMM), colonies consisting of �50 cells were scoredwith an inverted microscope. The effect of dCyd and/or AZT on
CFU-GM or CFU-GEMM colony growth was expressed as thepercentage of colony formation by drug-treated cells relative to theuntreated controls. The experiments were performed in duplicate
and repeated at beast four times for each condition tested.Intracellular AZT-TP, dTTP, and dCTP levels. Nonadherent
BMMC were isolated as described above, and suspended in McCoy’s
5a medium containing 10% FCS at a cell density of 106 cells/mL.Alternatively, HUT-102 cells were used in suspension culture inRPMI 1640 medium with 5% FBS. The two cell types in suspensionwere exposed to the designated concentrations of AZT and/or dCyd
for 6 hours in a 37#{176}C,5% CO2 incubator. After the incubation, cellswere pelleted and neutralized and perchboric acid soluble extracts
were obtained as described previously.27 The acid soluble extractswere treated with periodate oxidation to remove endogenous ribonu-
cleotides.25 AZT-TP, dCTP, and dTTP were quantitated by a minormodification of a high pressure liquid chromatography method.2028Briefly, a radial pak SAX anion exchange column (Waters, Milford,MA) was used with a 350 mmol/L ammonium phosphate buffer(pH 3.8) as the mobile phase at a 2.5 mL/min flow rate. Absorbance
at 280 nm was quantitated with the aid of a dual wavelength
absorbance detector (Waters). dCTP, dTTP, and AZT-TP wereidentified by co-elution with known standards and peak height ratios
at 254 and 280 nm. Values are expressed as pmol/ 106 cells ± SEM.AZT incorporation into DNA. After nonadherent BMMC were
isolated, washed, and suspended as described above, they were
placed in T-flasks containing 10 gzmol/L of [3H] AZT ± dCyd (100
gzmol/L or 1 mmol/L). Alternatively, HUT-I02 cells were used insuspension culture in RPMI 1640 medium with 5% FBS. The flasks
were incubated for 6 hours, after which the suspension was trans-
ferred to 50-mL tubes and centrifuged at 400 g for 8 minutes at 4#{176}C.
The cell pellet was washed twice with cold PBS to remove theradiolabeled drug. DNA was isolated and purified by proteinase K
and RNAase digestion, phenol/chloroform extraction, and ethanol
precipitation as previously described.� After centrifugation at
12,000 g at 4#{176}Cfor 30 minutes, the DNA was resuspended in
Tris-HCL buffer, quantitated spectrophotometrically, and aliquotswere removed and placed in scintillation vials to determine theradioactivity. Values for each condition are expressed as fmol/gzg of
DNA.
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DEOXYCYrIDINE REDUCES Air CYTOTOXICITY 1925
Table 1 . Effect of dCyd on AZT-Mediated Inhibition of Colony
Growth of CFU-GM and CFU-GEMM
Conditions
Control of Colon y Growth (%)
CFU-GM CFU-GEMM
AZT 1Og�mol/L 30.9 ± 3.1 31.7 ± 7.0
AZT 10 �amol/L + dCyd 10 g.amol/L 47.6 ± 2.9 48.5 ± 2.5
AZT 10 MmoI/L + dCyd 1 mmol/L 80.0 ± 5.0’ ND
AZT100�zmol/L 6.3± 1.5 11.9 ±3.7
AZT 100 �imoI/L + dCyd 100 Mmol/L 33.9 ± 4.4 ND
AZT 100 gzmol/L + dCyd 1 mmol/L 55.7 ± 4.5 53.7 ± 9.0
Normal BMMC from four or more healthy volunteers were plated in
soft agar (CFU-GM) or methylcellulose (CFU-GEMM) in the continuous
presence of AZT and/or dCyd. After 7 days (CFU-GM) or 1 4 days
(CFU-GEMM), colonies consisting of 50 or more cells were scored with
an inverted microscope. Values represent the percentage of control
colony formation ± SEM.
Abbreviation: ND, not done.
#{149}Significantly different (P < .05) from colony growth in the presence
of identical concentration of AZT administered alone.
Statistical analysis. Significant differences between experi-mental groups were assessed using Student’s t test for paired
observation.
RESULTS
The protective effect of dCyd on AZT-mediated incuba-
tion of soft agar colony growth of normal CFU-GM and
CFU-GEMM is shown in Table I . The range of control
colony growth for normal CFU-GM and CFU-GEMM was
30 to 149 colonies and 20 to 60 colonies/well, respectively.
Continuous exposure to AZT (10 �tmob/L) alone resulted in
30.9% and 31.7% of control CFU-GM and CFU-GEMM
colony growth, respectively. Simultaneous exposure to equi-
molar concentration (10 �amob/L) ofdCyd and AZT resulted
in a significant protection of colony growth for CFU-GM
(46.6 ± 2.9) as well as CFU-GEMM (48.5 ± 2.5). Co-
administration of 100 �tmol/L dCyd together with AZT (10
�tmol/L) restored colony formation by CFU-GM to 7 1.1%
and by CFU-GEMM to 64.8%. In the case of CFU-GM, a
further improvement in colony growth was obtained by
administering I mmol/L dCyd in conjunction with 10 �amol/
L AZT. At higher level of AZT (100 �tmol/L), a greater
reduction in CFU-GM and CFU-GEMM colony formation
was observed. However, once again, coculture with I mmob/
L dCyd exerted a significant protection toward the bone
marrow progenitor cells in semisolid culture (Table 1).
The effect of dCyd on the biochemical perturbations by
AZT (1 0 zmob/L) in normal BMMC and HUT- 102 cells are
depicted in Tables 2 and 3, respectively. Administration of
100 �zmol/L or 1 mmol/L dCyd for 6 hours to normal
BMMC significantly expanded dCTP and dTTP pools-
approximately a twofold expansion on exposure to I mmol/L
dCyd (Table 2). Exposure of BMMC to 10 �smol/L AZT for
6 hours reduced the intracellular dCTP bevels from 5.7 to 0.9
pmol/ 1 06 cells and dTTP bevels from 6.2 to I .3 pmol/ 106,
along with a significant accumulation of AZT-TP pools
(20.2 pmol/l06 cells). Simultaneous exposure of normal
BMMC to 10 �imol/L AZT and 100 �imob/L dCyd com-
pletely restored the intracellular dCTP and dTTP pools to
the control values and significantly reduced AZT-TP bevels
(from 20.2 to 2.7 pmob/106 cells). When the cells were
exposed to I mmol/L dCyd, the intracellular AZT-TP
generation was further reduced to undetectable levels (Table
2). In HUT-102 cells, AZT (10 g�mob/L) induced reduction
in dCTP bevels was not as profound as seen in normal
BMMC. Also, dCyd mediated expansion of intracellular
dCTP and dTTP pools in the presence or absence of AZT (10
�mol/L) was not significantly different from what was
observed in BMMC (Tables 2 and 3). However, dCyd
exerted a significantly reduced effect on the accumulation of
AZT-TP in HUT-102 cells versus BMMC treated with 10
�tmol/L AZT. DNA incorporation of 10 �imol/L [3H] AZT
was 22.5 and 62.1 fmob/g.�g in normal BMMC versus HUT-
102 cells. In the presence of 100 gzmol/L and I mmol/L
dCyd, [3H] AZT DNA incorporation was reduced to 4.9 and
2.6 fmol/g�g in normal BMMC, respectively; corresponding
values in HUT-I02 cells were 35.2 and 23.7 fmol/�ig,
respectively.
Table 4 shows the effect of dCyd on the ability of AZT to
suppress HIV infection and cytotoxicity in HUT-102 cells.
When these cells were exposed to an HIV containing super-
natant of an infected clone of CEM cells, high levels of p24
antigen (63.0 x iO� pg/mL) could be assayed in the culture
supernatant at the end of 7 days and 86% of the cells
expressed HIV antigens, as determined by indirect immuno-
fluorescence. Only I 5.0% of the cells were viable at the end
of 7 days. From the third to the seventh day, the cell free
supernatant from the culture showed an increment in the p24
Table 2. dCTP. dTTP. and AZT-TP Accumu lation in Normal Bone Ma rrow Mononuclear Cells Exposed to dCyd and/or AZT
Conditions
Concentrations (pmol/ 10� Cells)
dCTP dTTP AZT-TP
Control
dCyd iOO gzmol/L
dCyd 1 mmol/L
AZT i0gzmol/L
AZT 10 gimol/L + dCyd 100 �zmoI/L
Air 10 gzmol/L + dCyd i mmol/L
5.7 ± 1.1
8.9 ± 1 .5
il.1 ± 1.9
0.9 ± 0.3
7.7 ± 2.2
7.5 ± 2.2
6.2 ± 1.7
9.3 ± 3.2
14.4 ± 6.2
1.3 ± 0.5
6.7 ± 3. 1
iO.5 ± 3.0’
-
-
-
20.2 ± 5.3
2.7 ± 1 .2
UD
Normal BMMC cells were exposed to the designated concentrations of AZT and/or dCyd for 6 hours. The cells were pelleted, and neutralized
perchloric acid extracts were obtained. Subsequently, the periodated extracts were analyzed for dCTP. dTTP, and AZT-TP pools by an HPLC method, as
described in the text. Values are expressed as pmol/10#{176} cells and represent the mean of six or more separate samples ± SEM.
Abbreviation: UD, undetectable [ie, below the level of detectability (��0.5 pmol/ 1 �8 cells) by HPLC method].
Significantly different (P < .05) from the corresponding triphosphate pools in the cells exposed to AZT alone.
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1926 BHALLA ET AL
Table 3. dCTP, dTTP, and AZT -TP Accumulation in HUT -102 Cells Exposed to dCyd and/or AZT
Conditions
Concentrations (pmol/ 1 0 Cells)
dCTP dTTP AZT-TP
Control
dCyd 1 00 gzmol/L
dCyd 1 mmol/L
AZT lOgimol/L
AZT 10 gimol/L + dCyd 100 gzmol/L
AZT lOgimol/L + dCyd 1 mmol/L
5.6 ± 0.4
7.8 ± 1 .2
10.0 ± 1.0
2.0 ± 0.3
4.3 ± 0. 1
5.8 ± 0.4
8.1 ± 1.3
1 1.0 ± 2.6
13.8 ± 3.3
1.5 ± 0.4
5.8 ± 1 .6
7.4 ± 1.3
-
-#{149}
-
35.8 ± 7.4
14.0 ± 1 . 1 �
2.5 ± 0.4
HUT- 1 02 cells were exposed to the designated concentrations of AZT and/or dCyd for 6 hours. The cells were pelleted, and neutralized perchloric acid
extracts were obtained. Subsequently, the periodated extracts were analyzed for dCTP, dTTP, and AZT-TP pools by an HPLC method, as described in the
text. Values are expressed as pmol/10 cells and represent the mean of six or more separate samples ± SEM.
Significantly different (P < .05) from corresponding triphosphate pools in normal BMMC (Table 2) treated identically.
antigen levels from 3.5 to 63.0 x iO� pg/mL. Exposure of the
cells to HIV and AZT (1 or 10 �tmol/L) for 7 days
profoundly reduced p24 antigen levels in the supernatant to
0.2 x l0� pg/mL, completely suppressed the cellular HIV
antigen expression, and improved cell viability. Co-incuba-
tion of the HIV-infected cells with AZT (1 or 10 gimol/L)
and dCyd (100 gsmob/L) did not significantly antagonize the
anti-HIV effect of AZT, as demonstrated by a persistently
low p24 antigen production and the absence of detectable
immunofluorescence. Also, the improvement in the viability
of l-IUT-102 cells caused by AZT was not altered by
co-administration of dCyd (Table 4). Administration of 1
mmol/L dCyd in conjunction with 10 gsmol/L AZT for 7
days resulted in 8.0% of the cells expressing HIV antigens by
immunofluorescence assay and a slight increase in the p24
levels (0.9 x l0� pg/mL) in the culture supernatant. At the
end of this incubation, the percentage of viable cells had not
changed significantly (42%).
DISCUSSION
Administration of high doses of AZT results in plasma
levels in the range of 5 to 10 gzmob/L, which are associated
with a significant suppression of HIV infectivity in patients
with AIDS.6 However, these levels of AZT produce marked
inhibition of in vitro growth of bone marrow progenitor cells
and are clinically associated with neutropenia and anemia,
particularly in patients with advanced AIDS.�’#{176} The results
of our in vitro study demonstrate that high but clinically
achievable and nontoxic concentrations of dCyd (�I00
�tmol/L)’5 can significantly reverse CFU-GM and CFU-
GEMM growth inhibition caused by high bevels of AZT
without impairing its anti-I-IIV activity.
The dose limiting bone marrow toxicity of AZT has been
largely attributed to the depletion of intracellular dCTP and
dTTP bevels.’0”2 In normal human BMMC exposed to high
levels of AZT (10 gzmol/L), intracellular dCTP and dTTP
pools decline with a concomitant generation of AZT-TP
pools.’2 In contrast, in cultured cell lines (HL-60, K-562, 1-19,
and MOLT-4) exposed to AZT, intracellular dCTP levels
have been demonstrated to increase.’3’30 This discrepancy in
the metabolic effects of AZT between freshly procured,
normal BMMC and cultured cell lines might be due to
significant differences in the activity of the salvage pathway
enzymes for nucleoside metabolism, or due to the differences
in the bevels of regulatory intracellular deoxyribonucbeotide
pools. A previous report3’ and our data in HUT-102 cells
highlight these differences in different cell types. As shown
in previous studies, administration of dCyd expands intracel-
bular dCTP levels in BMMC.20’2’ Furthermore, deamination
of dCMP to dUMP and its subsequent conversion to
dTMP’7”8 might overcome the competitive substrate inhibi-
tion ofdTMP kinase by AZT-MP2 (Fig I), and would result
in expansion of intracellular dTTP pools in cells treated with
AZT plus dCyd. Also, since thymidine kinase is subject to
feedback inhibition by dTTP,’8 expansion of intracellular
Table 4. Effect of dCyd and AZT on HIV In fectivity and Cytotoxicity in HUT-i 02 Cells
Immunoflucrescence HIV p24 Antigen Cell Viability
Conditions
1% Positive Cells)
Day 3 Day 7
(pg/mL) (% of Control)
Day 7Day 3 Day 7
HlVsupernatant(1 mL) 35.0 86.0 3.5 x iO� 63.0 x i0� 15.0
HIV + AZT 1 gimol/L 0 0 0.5 x i0� 0.2 x i0� 75.0
HIV + AZT 1 gimol/L +
dCyd 100 gimol/L 0 0.5 0.6 x iO� 0.8 x i0� 71.0
HIV + Air lOgimol/L 0 0 0.4 x i0� 0.2 x i0� 44.0
HIV + AZT lOgimol/L
+ dCyd 100 gimol/L 0 0 0.7 x i0� 0.3 x i0� 49.0
HIV + AZT l0gimol/L
+ dCyd 1 mmol/L 0.2 8.0 0.6 x i0� 0.9 x iO� 42.0
Uninfected and HIV-infected HUT- 1 02 cells were incubated with the designated concentrations of AZT with or without dCyd. At the end of 3 or 7
days. samples were taken for the determination of cell viability and HIV antigen expression by immunofluorescence, as described under Methods. HIV
p24 antigen was assayed in the supernatant fluids from all culture conditions utilizing Coulter enzyme immunoassay. The values represent the means of
two experiments performed as duplicate conditions.
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REFERENCES
DEOXYCYTIDINE REDUCES AZT CYTOTOXICITY 1927
dTTP pools would reduce AZT-TP levels. In BMMC treated
with AZT and dCyd, we also observed a concomitant reduc-
tion in DNA incorporation of [3H] AZT, most likely because
the mammalian DNA polymerase alpha has a greater affin-
ity for the natural substrates dCTP and dTTP as compared
with AZT-TP.2 Additionally, in our study, the generation of
AZT-TP pools in nonadherent BMMC, not surprisingly, is a
different finding from the one showing inefficient phospho-
rybation of AZT in terminably differentiated, nondividing,
primary human monocyte derived macrophages.32 The
BMMC population separated by density grandient centrifu-
gation and depleted of adherent cells is heterogenous in
nature and does not represent a pure population of progenitor
cells. Therefore, the results of our biochemical studies in
BMMC may not accurately reflect the biochemical pertur-
bations in a pure progenitor cell population. It has not yet
been possible to sufficiently purify large numbers of cbono-
genic bone marrow progenitor cells to determine the intracel-
lular biochemical effects of AZT and/or dCyd in these
cells.
Our studies in HIV-infected HUT-102 cells highlight the
ability of high levels of AZT to suppress HIV infection, even
in the presence of high concentrations of dCyd. This phenom-
enon might be partly explained by the significantly higher
generation of AZT-TP or dl’TP pool ratios in HUT-102 cells
versus BMMC when the two cell types are co-incubated with
AZT and dCyd. A previously published report has also
suggested that the intracellular ratio of the dideoxynucbeo-
side triphosphate to normal deoxynucleoside triphosphate
may be a crucial factor in determining the activity of
dideoxynucleosides against HIV.3’ Higher AZT-TP to dTTP
pool ratios in HUT-102 cells treated with [3H] AZT plus
dCyd might also explain higher DNA incorporation of AZT
in HUT-102 cells. Previous studies have also found striking
differences in the metabolism and activity of AZT in
different cell species.33 Although it would have been more
pertinent to have examined the effects of dCyd on the
1 . Yarchoan R, Broder 5: Development of antiretroviral therapyfor the acquired immunodeficiency syndrome and related disorders.N EngI i Med 316:557, 1987
2. Furman PA, Fyfe JA, St. Clair MH, Weinhold K, Rideout JL,Freeman GA, Lehrman SN, Bobognesi DP, Broder S, Mitsuya H,Barry DW: Phosphorylation of 3’azido-3’-deoxythymidine and selec-
tive interaction of the 5’-triphosphate with human immunodefi-ciency virus reverse transcriptase. Proc Nail Acad Sci USA 83:8333,1986
3. DeVita VT, Broder S. Fauci AS, Kovacs iA, Chabner BA:
Developmental therapeutics and the acquired immunodeficiencysyndrome. Ann Intern Med 106:568, 1987
4. Mitsuya H, Weinhold KJ, Furman PA, St. Clair MH, Lehr-man SN, Galbo RC, Bolognesi D, Barry DW, Broder S: 3’-azido-3’-deoxythymidine (BWA5094): An antiviral agent that inhibits theactivity and cytopathic effect of human T-lymphotropic virus typeIll/lymphadenopathy-associated virus in vitro. Proc NatI Aced Sci
USA 82:7096, 1985
5. Fischb MA, Richman DD, Grieco MH, Gottlieb MS. Volberd-ing PA, Laskin OL, Leedom JM, Groopman JE, Mildvan D,Schooley RT, Jackson GG, Durack DT, King D: The efficacy of
metabolism and anti-HIV activity of AZT in HIV-infected
bone marrow progenitor cells, an in vitro model system that
would permit these biochemical studies is not yet available.
Our data indicate that AZT improves the viability of
HIV-infected HUT-102 cells, and co-administration of dCyd
and AZT maintains this improvement in cell viability with-
out antagonizing the anti-HIV activity of AZT. The defini-
tive mechanism underlying this differential effect of dCyd
remains to be elucidated. Nevertheless, in the presence of
dCyd, the ratio of intracellular concentration of AZT-TP to
its Ki value for HIV reverse transcriptase (RT) might
remain sufficiently high to inhibit HIV RT in HIV-infected
HUT-102 cells.34 But the ratio of AZT-TP to its Ki for DNA
polymerase alpha of HUT-l02 cells might be significantly
reduced by dCyd to account for its differential effect on the
cytotoxicity of AZT.34
Several pharmacologic strategies are being explored to
improve the chemotherapeutic selectivity of AZT. Recombi-
nant granubocyte macrophage colony stimulating factor and
erythropoietin have been demonstrated to reduce the bone
marrow toxicity of AZT in vitro.8”2 Naturally occurring
nucleosides such as thymidine and uridine have also been
shown to have such beneficial effects.8’” However, thymidine
interferes with the anti-HIV activity of AZT,4’35 and higher
concentrations of thymidine (�lO smol/L) have growth
inhibitory effects on bone marrow progenitor cells.36
Although uridine does not interfere with the anti-HIV
activity of AZT35 a continuous exposure to high concentra-
tions of uridine has attendant toxicity.37 The results of the
present in vitro study indicate that high but nontoxic levels of
dCyd combined with continuously administered high concen-
trations of AZT, which optimally inhibit HIV replication4
and have significant clinical efficacy,� result in enhance-
ment of the chemotherapeutic selectivity of AZT. These
findings have potential implications for designing future
drug regimens incorporating AZT and dCyd with the poten-
tiab for an improved therapeutic index.
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1989 74: 1923-1928
K Bhalla, M Birkhofer, GR Li, S Grant, W MacLaughlin, J Cole, G Graham and DJ Volsky preservation of antiretroviral activityvitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with 2'-Deoxycytidine protects normal human bone marrow progenitor cells in
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