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The effect of 5-bromodeoxyuridine on human chromosomes
Experimental
182
PRELIMINARY THE
EFFECT
Department
M.
KABACK,’
of Pediatrics,
Uniuersiry
E.
CHROMOSOMES SAKSKLA of Pennsylvania
Received
34, 182-212
(19G4)
NOTES
OF 5-BROMODEOXYIJRIDINE HUMAN
M.
Cell Research
August
and
W.
and The
ON
l J.
hlELLMAri3
Wietar
Institute,
Pa.,
U.S.A.
5, 1963
Hse
and s omers 1.51have reported that 3 non-human mammalian cell lines grown in the presence of 5bromodeoxyuridine (RUDI~) show scvcral striking and specific alterations of chromosome morphology. HUI)It, a specific thymidine analoguc, causes marked lcnglhening of centromcric regions and secondary conslriclion areas, as well as an increase in the Frequency of chromatid breakage especially at sites of secondary constrictions. \Ye have studied the effecl of HUUI~ on human chromosomes of cultured pcripheral blood cells in an attempt to determine whether similar effects vn the human karyotype could be demonslratcd. Material und mpfhods. Culture of human lcukocytcs (Type 0, healthy adult male) were prepared according to the method of Moorhead et nl. [6] except that thgmine was omitted from the medium TC-199 used and the polymorphonuclcar leukocytes wcrc rernovcd 121. Removal of polymorphonuclcar leukocytes provided a more uniform inoculum of mononuclear cells and avoids their serving as a possiblethymine source when they dcgcnerate in the culture. I
Cell
Research
34
Contract Training University
Ko. 13-K-157. Program under of Pennsylvania,
a
Effect 01 Q-bromodeoxyuridine
Fig. 1. a S-Ii /Ig~nll sorncs
on humnn
chromosomes
183
from (‘LOO .omo2031.
groups, revealed that no grosschromosomal rearrangemenls or allerations in numht!r had occurred with BKDR treatment. It appeared, however, that secondary constrictions vvcre intensified and thereby more frequenlly observed in the chromosomes of HLIDR-treated cells. Therefore all analyzed cells were scored for the presence and intensity of secondary constriclions. The intensified secondary constrictions appeared as narrowed and noticeably stretched segments of chromatin. They were particularly evident in the subcenlromcric region of the $1 chromosomes and in the proximal part of the long arm of one of the chromosome pairs of the 6-12 group (Fig. 1). The constriction in this latter chromosome has been described and its presence used to designate -the $5) chromosome [ 1, 81. There was considerable variation in the intensity of the secondary constriction in chromosomes $1 and $9 ranging from no apparent constriction to a severely strelched segment of chromalin (I:&. 2). It can readily be seen that both the frequency and degree of enhancement of the secondary constriction in chromosomes Rrperimenlal
Cell
Ilcsrnrch
34
Xi. M. Kabuck, E. Snksela rend W. J. Mellman
Fig. 2.- Secondary wnslrictions of the $1 and $9 chromosomes in 4 represcnlativc RUI)Htreated cells (A through D) as compared wiLh those seen in 4 untrealed cells (E Lhrough H). Strong secondary constriclions are seen in boLh ft chromosomes of Cell A and in the left homologuc of Cell C. 7‘11~ #l’s of Cell I) shovv only a slight constriclion effect. Slight constrictions arc also seen in the +1’s of the unLreated (;rll E while those of I;, G, and H show no secondary conslrictioris. All Ihe #9’s of the 4 treated cells (A through I)) contain strong secondary constrictions. In the untreated cells, Lhe 99’s in Cell F and the right hornologue of Cell H show modrralc constrictions while the two in Cell E show only slight effect.
#l and $l were significantly increased by RI!I)H treatment (Table I). The incidence ol severe stretching (“strong elf&“) in the treated cells is increased approximately five-fold while the overall frequency of the secondary constriction is increased by 100 per cent for #I and approximately 50 per cent for $0. With HUDH treatment two other secondary constrictions showed an apparent increase in frequency. One of the two secondary constrictions in a B-X group chromosome [8] (middle of the long arm) was seen more commonly among treated cells. This was also the cast for the constriction in the $16 chromosome (subccntromeric region of the long arm) (Table II). Neither of these constrictions, however, showed the stretched and narrowed effect seen in the $1 and 89. Rather they appeared as short segments of undcrstained chromatin of a more hazy and puffy nature. Of the 300 cells analyzed all except 5 contained 46 chromosomes. These 5 cells were hypodiploid with no consistency of the missing chromosomes, and were divided between treated (three) and control (two) groups. Lengthening of centromcric regions, chromatid breaks, or apparent effect on other areas previously described with secondary constrictions 13, 4, 81 were not evident after BUI)I~ treatment. Experimenlal
Cell Hesearch
34
Effect of 54romodeozyuridine
on human
chromosomes
185
T>iscussion.--From these studies it appears thal TS1:I)R has a specific action on certain secondary constrictions of human chromosomes. In chromosomes $1 and $9 both the frequency and intensity of the secondary constriction areas were markedly increased. It is noted that the nature of enhancement of these constrictions with HI:DR is considerably different from that reported with modified fixation procedures, [8] or with pretreatment in calcium-free medium [9[. Although the observed secondary constriction in a 6-X group chromosome and the one in the $16 chromosome was more commonly seen after HI: I)H treatment, this degree of enhancement was inconclusive in light of its low frequency. Evidence including the data presented here that nucleic acid analogucs or nuclcotitle antagonists may have morphologic effects on specific chromosomes [lo] suggests a potential technique for the labelling and identification of specific human rhromosomes and chromosome fragments. UTe have not detcrmincd if the so-called hcterochromatic X is affected by 13UDH treatment, since only XY cells were used in these studies. Since peripheral blood cells have been shown to undergo one or at most two mitotic divisions in typical
'I‘J\n~~
I.
of the
Cornprison
sfrictions
seen
in
frequency
ChrOmOSOmeS rtnireated
Snmbers
and no.
peripheral
refer Lo the number eflects, the most
degree
1 and
of
no.
blood
enhancement
9 from
Cell
CJ~ secondar!y
Ti'C'DH-treated
CUlillres.
of cells. Where homologues showed affected chromosome was scored. (komosome
con-
and
no. 1
different
Oromosomc
no. Y
-_
5-IsuI)r~-
5-HUI)RLrealcd
Intensity of secondarv constriction
Sonc Slight ~iodcrate Strong
SCC?II
Total
TABLE
cells
II.
Frequency
of
secondarg
No. so. so. No.
1 6-x 0 16
r--hc BlTDR
llntreated
1.50 150 1.50 1.50
1.X 150 1.X 150
Lrea ted
Untreated
GY 22 23 36
112 18 13 ‘i
51 16 27 53
86 3.5 18 11
150
150
15!,
150
consfriciions
So. cells
Chromosome
Cnlrcated
in
H C’I)R-treated
No. Cells showing secondary constriction 7 (; I3UDH IInLreaLed 81 10 96 18
and
untreated
cells.
Frequency of secondary constriction C BI;DH
Untreated
51 6.5 64 12
25.3 1.3 42.7 3.3
38 2 64 5 Experirnrnld
‘$ :a :i, y: Cdl
Rrsetrrch
‘S;, I;0 “b y? 34
E. W. Rice
186
s-day cullures 11, 71 it is also possible that other chromosomal effects might be produced if the leukocytes were exposed to the agent during multiple mitotic cycles. Summwy---IHuman peripheral blood leukocytes were cultured for 3 days in lhc presence of 5-bromodeoxyuridine (ZNO pug/ml). This treatment effected a specific increase in the frequency and intensity of secondary constrictions of chromosome $1 and //Cl without otherwise altering chromosome morphology or -Lhe karyotype. The authors thank I.)r P. S. Moorhead for his advice during this investigation and for his review of lhis manuscript. REFEREXCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
HEXDI~, Xl. A. and PFWSCOTT, D. .\I., E.zpll Cell Iles. 27, 221 (19G2). (1962). CooPER, II. I.. and 1hRSC11110RN, I<., Hlood 20, 101 Abslract 1;F.I~(il:sos-sYI1.11, $1. A., hoc. Royd. sot. Med. 55, 451 (1962). FORD, C. E., J. ~Ynll Cnncer Inst. Monograph So. 7 (1962). Hsu, T. C. and SOMERS, C. J<., I’roc. ,\‘al/ Acad. Sci. 47, 396 (1961). MOORHEAD, P. S., SOWEI.~., P. C.: ?JELI,MIAN, W. .J,: BATTIPS, I). M., and II~JSGEIWOI~~, Exptl Cdl Res. 20, 61.7 (1960). PRESCOTT, 11. JI. and BESTIER, 31. I\., Ezpff Cell fI. S. and MAKISO, S., Am. J. flum. Gen. 15, 24 (1963). SOMEHS, C. K. and Hsc, T. C., Proc. .Tal! Acnd. Sci. 48, 9.75 (1962).
MORPHOLOGTCAJ~ FOLLOWING
CHA_R;GES
TREATMENT
IN
HUMAIV
OF SEMEN
1.1. A.,
SPEKMATOZOA WITH
CERTAI?i
DTAT,KYI,DITHIOC~\l~Bi\~~ATES’ F;. W. R I C I’ William
1.1. Singer General
Memorial Hospital, Recbed
Reseurch Laboratory of the Allegheny Pittsburgh, l’u., U.S. A.
September 13, 1963
I-x a recent sludy of Holzaepfcl cl al. on the evaluation of the spermicidal effcctiveness of a group of 581 organic compounds, the sodium sails of dimethyl and dicthyl dilhiocarbamate and the dimethyl ammonium sail of dimethgl dithiocarbamatc showed the highest activity- of all chemicals tested [Il. In addition to confirming the marked spermicitlal activity of these three dithiocarbamates il has been observed furthermore that sodium diethyl dithiocarbamate and diethgl ammonium dimethy1 dithiocarbamnte may produce on the tails of human spermatozoa, initially either viable or non-viable, gross morphological alterations, consisting of an in1 A rcpnrl of this invesligation Federation of Amcricnn Socielies 16 -20, 1963.
was presealcd for Expwimenlal
al
the Forty-seventh Lliology, A.llantic
Annual hleeling Cily, Sew Jersey,
of the April
182
PRELIMINARY THE
EFFECT
Department
M.
KABACK,’
of Pediatrics,
Uniuersiry
E.
CHROMOSOMES SAKSKLA of Pennsylvania
Received
34, 182-212
(19G4)
NOTES
OF 5-BROMODEOXYIJRIDINE HUMAN
M.
Cell Research
August
and
W.
and The
ON
l J.
hlELLMAri3
Wietar
Institute,
Pa.,
U.S.A.
5, 1963
Hse
and s omers 1.51have reported that 3 non-human mammalian cell lines grown in the presence of 5bromodeoxyuridine (RUDI~) show scvcral striking and specific alterations of chromosome morphology. HUI)It, a specific thymidine analoguc, causes marked lcnglhening of centromcric regions and secondary conslriclion areas, as well as an increase in the Frequency of chromatid breakage especially at sites of secondary constrictions. \Ye have studied the effecl of HUUI~ on human chromosomes of cultured pcripheral blood cells in an attempt to determine whether similar effects vn the human karyotype could be demonslratcd. Material und mpfhods. Culture of human lcukocytcs (Type 0, healthy adult male) were prepared according to the method of Moorhead et nl. [6] except that thgmine was omitted from the medium TC-199 used and the polymorphonuclcar leukocytes wcrc rernovcd 121. Removal of polymorphonuclcar leukocytes provided a more uniform inoculum of mononuclear cells and avoids their serving as a possiblethymine source when they dcgcnerate in the culture. I
Cell
Research
34
Contract Training University
Ko. 13-K-157. Program under of Pennsylvania,
a
Effect 01 Q-bromodeoxyuridine
Fig. 1. a S-Ii /Ig~nll sorncs
on humnn
chromosomes
183
from (‘LOO .omo2031.
groups, revealed that no grosschromosomal rearrangemenls or allerations in numht!r had occurred with BKDR treatment. It appeared, however, that secondary constrictions vvcre intensified and thereby more frequenlly observed in the chromosomes of HLIDR-treated cells. Therefore all analyzed cells were scored for the presence and intensity of secondary constriclions. The intensified secondary constrictions appeared as narrowed and noticeably stretched segments of chromatin. They were particularly evident in the subcenlromcric region of the $1 chromosomes and in the proximal part of the long arm of one of the chromosome pairs of the 6-12 group (Fig. 1). The constriction in this latter chromosome has been described and its presence used to designate -the $5) chromosome [ 1, 81. There was considerable variation in the intensity of the secondary constriction in chromosomes $1 and $9 ranging from no apparent constriction to a severely strelched segment of chromalin (I:&. 2). It can readily be seen that both the frequency and degree of enhancement of the secondary constriction in chromosomes Rrperimenlal
Cell
Ilcsrnrch
34
Xi. M. Kabuck, E. Snksela rend W. J. Mellman
Fig. 2.- Secondary wnslrictions of the $1 and $9 chromosomes in 4 represcnlativc RUI)Htreated cells (A through D) as compared wiLh those seen in 4 untrealed cells (E Lhrough H). Strong secondary constriclions are seen in boLh ft chromosomes of Cell A and in the left homologuc of Cell C. 7‘11~ #l’s of Cell I) shovv only a slight constriclion effect. Slight constrictions arc also seen in the +1’s of the unLreated (;rll E while those of I;, G, and H show no secondary conslrictioris. All Ihe #9’s of the 4 treated cells (A through I)) contain strong secondary constrictions. In the untreated cells, Lhe 99’s in Cell F and the right hornologue of Cell H show modrralc constrictions while the two in Cell E show only slight effect.
#l and $l were significantly increased by RI!I)H treatment (Table I). The incidence ol severe stretching (“strong elf&“) in the treated cells is increased approximately five-fold while the overall frequency of the secondary constriction is increased by 100 per cent for #I and approximately 50 per cent for $0. With HUDH treatment two other secondary constrictions showed an apparent increase in frequency. One of the two secondary constrictions in a B-X group chromosome [8] (middle of the long arm) was seen more commonly among treated cells. This was also the cast for the constriction in the $16 chromosome (subccntromeric region of the long arm) (Table II). Neither of these constrictions, however, showed the stretched and narrowed effect seen in the $1 and 89. Rather they appeared as short segments of undcrstained chromatin of a more hazy and puffy nature. Of the 300 cells analyzed all except 5 contained 46 chromosomes. These 5 cells were hypodiploid with no consistency of the missing chromosomes, and were divided between treated (three) and control (two) groups. Lengthening of centromcric regions, chromatid breaks, or apparent effect on other areas previously described with secondary constrictions 13, 4, 81 were not evident after BUI)I~ treatment. Experimenlal
Cell Hesearch
34
Effect of 54romodeozyuridine
on human
chromosomes
185
T>iscussion.--From these studies it appears thal TS1:I)R has a specific action on certain secondary constrictions of human chromosomes. In chromosomes $1 and $9 both the frequency and intensity of the secondary constriction areas were markedly increased. It is noted that the nature of enhancement of these constrictions with HI:DR is considerably different from that reported with modified fixation procedures, [8] or with pretreatment in calcium-free medium [9[. Although the observed secondary constriction in a 6-X group chromosome and the one in the $16 chromosome was more commonly seen after HI: I)H treatment, this degree of enhancement was inconclusive in light of its low frequency. Evidence including the data presented here that nucleic acid analogucs or nuclcotitle antagonists may have morphologic effects on specific chromosomes [lo] suggests a potential technique for the labelling and identification of specific human rhromosomes and chromosome fragments. UTe have not detcrmincd if the so-called hcterochromatic X is affected by 13UDH treatment, since only XY cells were used in these studies. Since peripheral blood cells have been shown to undergo one or at most two mitotic divisions in typical
'I‘J\n~~
I.
of the
Cornprison
sfrictions
seen
in
frequency
ChrOmOSOmeS rtnireated
Snmbers
and no.
peripheral
refer Lo the number eflects, the most
degree
1 and
of
no.
blood
enhancement
9 from
Cell
CJ~ secondar!y
Ti'C'DH-treated
CUlillres.
of cells. Where homologues showed affected chromosome was scored. (komosome
con-
and
no. 1
different
Oromosomc
no. Y
-_
5-IsuI)r~-
5-HUI)RLrealcd
Intensity of secondarv constriction
Sonc Slight ~iodcrate Strong
SCC?II
Total
TABLE
cells
II.
Frequency
of
secondarg
No. so. so. No.
1 6-x 0 16
r--hc BlTDR
llntreated
1.50 150 1.50 1.50
1.X 150 1.X 150
Lrea ted
Untreated
GY 22 23 36
112 18 13 ‘i
51 16 27 53
86 3.5 18 11
150
150
15!,
150
consfriciions
So. cells
Chromosome
Cnlrcated
in
H C’I)R-treated
No. Cells showing secondary constriction 7 (; I3UDH IInLreaLed 81 10 96 18
and
untreated
cells.
Frequency of secondary constriction C BI;DH
Untreated
51 6.5 64 12
25.3 1.3 42.7 3.3
38 2 64 5 Experirnrnld
‘$ :a :i, y: Cdl
Rrsetrrch
‘S;, I;0 “b y? 34
E. W. Rice
186
s-day cullures 11, 71 it is also possible that other chromosomal effects might be produced if the leukocytes were exposed to the agent during multiple mitotic cycles. Summwy---IHuman peripheral blood leukocytes were cultured for 3 days in lhc presence of 5-bromodeoxyuridine (ZNO pug/ml). This treatment effected a specific increase in the frequency and intensity of secondary constrictions of chromosome $1 and //Cl without otherwise altering chromosome morphology or -Lhe karyotype. The authors thank I.)r P. S. Moorhead for his advice during this investigation and for his review of lhis manuscript. REFEREXCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
HEXDI~, Xl. A. and PFWSCOTT, D. .\I., E.zpll Cell Iles. 27, 221 (19G2). (1962). CooPER, II. I.. and 1hRSC11110RN, I<., Hlood 20, 101 Abslract 1;F.I~(il:sos-sYI1.11, $1. A., hoc. Royd. sot. Med. 55, 451 (1962). FORD, C. E., J. ~Ynll Cnncer Inst. Monograph So. 7 (1962). Hsu, T. C. and SOMERS, C. J<., I’roc. ,\‘al/ Acad. Sci. 47, 396 (1961). MOORHEAD, P. S., SOWEI.~., P. C.: ?JELI,MIAN, W. .J,: BATTIPS, I). M., and II~JSGEIWOI~~, Exptl Cdl Res. 20, 61.7 (1960). PRESCOTT, 11. JI. and BESTIER, 31. I\., Ezpff Cell f
MORPHOLOGTCAJ~ FOLLOWING
CHA_R;GES
TREATMENT
IN
HUMAIV
OF SEMEN
1.1. A.,
SPEKMATOZOA WITH
CERTAI?i
DTAT,KYI,DITHIOC~\l~Bi\~~ATES’ F;. W. R I C I’ William
1.1. Singer General
Memorial Hospital, Recbed
Reseurch Laboratory of the Allegheny Pittsburgh, l’u., U.S. A.
September 13, 1963
I-x a recent sludy of Holzaepfcl cl al. on the evaluation of the spermicidal effcctiveness of a group of 581 organic compounds, the sodium sails of dimethyl and dicthyl dilhiocarbamate and the dimethyl ammonium sail of dimethgl dithiocarbamatc showed the highest activity- of all chemicals tested [Il. In addition to confirming the marked spermicitlal activity of these three dithiocarbamates il has been observed furthermore that sodium diethyl dithiocarbamate and diethgl ammonium dimethy1 dithiocarbamnte may produce on the tails of human spermatozoa, initially either viable or non-viable, gross morphological alterations, consisting of an in1 A rcpnrl of this invesligation Federation of Amcricnn Socielies 16 -20, 1963.
was presealcd for Expwimenlal
al
the Forty-seventh Lliology, A.llantic
Annual hleeling Cily, Sew Jersey,
of the April