The phenomenon of delayed disruption of telomeric links between chromosomes

Vladimir I. Stobetsky sto at aha.ru
Mon Mar 9 04:05:03 EST 1998


Key  words : CHROMOSOMA,INDUCED  TELOMERIC FUSION

The phenomenon of delayed disruption of telomeric links between
chromosomes

The history of phenomena  of mitotic chromosome transformation (MCT)
began in 1964 when the chromosome morphology  of Chinese hamster cell
line Don was described under prolonged colcemid treatment that induced
the cells with micronuclei. At the first mitotic division of these
polykaryocytes the chromosomes  with  portions of delayed spiralization
(PDS) were discovered (Stubblefield,1964).

The simultaneous treatment with colcemid and 5-bromodeoxyuridine
(5-BrdU)  of Chinese hamster cells led to appearance of dicentric
chromosomes at the first mitotic division of cells with micronuclei. The
absence of double fragments in such metaphases and the fact that they
could not be lost brought us  to the conclusion that dicentrics were
formed by  "end-to-end fusions  " (Stobetsky,1976). As the 5-BrdU was
incorporated into DNA for two  S-periods it was shown that chromosomes
in dicentrics were in the condition of telomeric fusion till the second
S-period. Therefore the discovered dicentrics were the result of delayed
disruption of telomeric links between chromosomes (Stobetsky,1978b). 

The phenomenon of delayed disruption of telomeric links between
chromosomes is the first  experimental model of the purposeful
influence on the function of telomeres in mammalian cells through MCT.
We used in our experiments the heteroploid Chinese hamster cell line
B11d-ii-FAF28 (clone237S) as a rule, but the phenomenon was   reproduced
in heteroploid Chinese hamster cell line CHO-6 also (Stobetsky,1990).
Another halogenated analogs of thymidine (HAT),except
5-fluorodeoxyuridine (5-FdU)  were applyed.

The chemicals, such as colcemid (0,1 microg/ml ), 5-BrdU (20 -50 microg/ml)
were
administrated in  actively growing cell culture, usually day after
seeding. Temperature of cell cultivation was 37_C  excepting  some
cases. The   cells   fixed  after   42   hours cultivation  with drugs.
About  30% metaphases of polykaryocytes contained dicentric chromosomes 
   
Some metaphases  contained besides  the normal chromosomes  also
chromosomes with PDS and  "chromosome pulverization".  The last
abnormalities were present in cell line B11d-ii-FAF28 (clone 237S)
after prolonged colcemid treatment alone (Stobetsky,1978a). Our
experiments  showed that chromosomes with PDS did not correspond  to
prematurely condensed chromosomes. Having analyzed  three cytogenetic
phenomena - delayed spiralization of metaphase chromosome portions,
delayed  disruption of telomeric links between chromosomes and premature
centromere division of  the X-chromosome - we concluded that in this
case the routine scheme (gene - protein - chromatin) could not work.
These phenomena are presumably associated with the existence of  a
system of MCT independent of the cytoplasm  (Stobetsky,1988c) .
	
It was shown in series of the experiments the following:

1.	The phenomenon of delayed disruption  of telomeric links between
chromosomes was reproduced in human-Chinese hamster cell hybrids
(Stobetsky et al.,1984).

2.	It was discovered that this phenomenon appeared when the 5-BrdU
was incorporated for two S-periods or during the first S-period , so the
differential incorporation of analog into sister chromatids was
necessary(Stobetsky, Mironova,1987).

3.	Another HAT such as 5-iododeoxyuridine (5-IdU) and
5-chlorodeoxyuridine (5-ChldU) also  induced telomeric chromosome fusion
in cells  with micronuclei derived from cell line B11d-ii-FAF28 (clone
237S) (Stobetsky et al.,1987;Stobetsky,Mironova,1988d).The intensity of
dicentric formation depends on molecular  weight of HAT. So the 5-BrdU
was more effective than 5-IdU,and 5-ChldU was more effective than
5-BrdU.Under subsequent incorporation of  5-IdU and 5-ChldU and vice
versa we found that strong or weak reaction in respect to dicentric
formation depends on which analog was administered in the first S-period
(Stobetsky, Mironova,1989).

4.	It was shown that certain factors exist which change the
intensity of dicentric formation. 5-azacytidine and hyperthermia  (40_
C)  enhanced the frequency of dicentric chromosomes when using 5-BrdU,
so that  sometimes tricentric  were formed (Stobetsky,Mironova,1988a;
Stobetsky,Mironova,1988b). In  the following experiments it was shown
that intensity of dicentric formation was directly connected  with the
temperature of cultivation when using 5-BrdU.The action of hyperthermia
(40_C)  during the whole  period (42 hrs) of colcemid and  5-BrdU
treatment or that  of  the hyperthermia (40_ C ) only for the first 17
hours ( the first interphase and the first mitosis) led to the increased
frequency of  dicentrics. Under condition of hypothermia (34_ C) the
frequency of dicentric formation decreases. Changes in cultivation
temperature during the last 25 hours of colcemid and 5-BrdU action (the
second interphase and the second mitosis)  exerted no influence on
dicentric formation frequency  (Stobetsky,1991a).

5.	The most significant data were obtained when Chinese hamster
cells B11d-ii-FAF28 (clone 237S) when exposed to colcemid,various HAT
and hyper-,normo and hypothermia. The minimum frequency of dicentrics
was found in the case of using  5-IdU and hypothermia (34_C) .Under
these conditions we observed the depression of mitotic activity and also
we often witnessed the chromosomes with PDS of good quality.The maximum
level of dicentrics was discovered under action of 5-ChldU and
hyperthermia  (40_ C).Chromosomes with PDS were not found  in the latter
case, but unusually  great frequency tri- and quadriradials was
registered  (Stobetsky,1991b). 

6.	Chinese hamster heteroploid  cell culture  was treated by
colcemid for 42 h ,of which within the first 17 h by 5-BrdU. Dicentric
chromosomes, that were formed at the 2nd  mitosis  after  colcemid
administration , were analysed using G-banding. Of 211 only 40 were
made  by  heterological   chromosomes.   Other 171 dicentrics fere
formed by telomeric fusion of autological  chromosomes. No correlation
was found between the length, centromeric index and frequency of
dicentric formation. (Stobetsky et al., 1994).   


References

1.	Stubblefield  E. (1964). DNA synthesis and chromosomal
morphology of Chinese hamster cells cultured in media containing
n-deacethyl-n-methylcolchicine (colcemid). In: Cytogenetics of cells in
culture. N.-Y. 223-248.

2.	Stobetsky V.I. (1976). Chromosome fusion by telomeres in cells
treated  with colcemid and 5-bromodeoxyuridine. (Russ).
Bull.Exptl.Biol.Med. No 9. 1142-1144.	

3.	Stobetsky V.I.  (1978a). Mammalian polykaryocytes in vitro. I. A
comparison of mitotic chromosome condensation in fused human
heterophasic cells and Chinese hamster cells with micronuclei .
Biologisches Zentralblatt .97: 587-594.

4.	Stobetsky V.I.  (1978b). Mammalian polykaryocytes in vitro. II.
The phenomenon of delayed disruption of telomeric links between
chromosomes.Biologisches  Zentralblatt . 97: 595-604.

5.	Stobetsky V.I., Grachev V.P., Mironova  L.L. (1984). The
phenomenon of  delayed disruption of telomeric links betveen chromosomes
in polykaryocytes from human-Chinese hamster cell hybrids. (Russ). No 7.
87-88.

6.	Stobetsky  V.I., Mironova  L.L. (1987).  A delayed disruption of
telomeric links between chromosomes in the experimentally induced cells
with micronuclei under treatment with 5-bromodeoxyuridine at the first
S-period after colcemid administration. (Russ). Tsitologia..
29:724-727.

7.	Stobetsky  V.I., Khapchaev Yu. Kh. ,Mironova  L.L. (1987). The
induction of delayed disruption of telomeric links between chromosomes
in cells with micronuclei by 5-iododeoxyuridine and 5-bromodeoxyuridine.
(Russ). Bull. Exptl. Biol. Med. No 7. 83-85.

8. 	Stobetsky V.I., Mironova L.L. (1988a). Effect of 5-azacytidine
on the number of polycentric chromosomes      in cells    with
micronuclei   during   exposure   to 5-bromodeoxyuridine. (Russ). Bull.
Exptl. Biol. Med. No4 479-480.
	
9.	Stobetsky V.I. ,Mironova  L.L.  (1988b). The enhanced intensity
of polycentric chromosome formation in cells with micronuclei after
5-bromodeoxyuridine treatment under prolonged hyperthermia at 40_C. 
(Russ). Tsitologia. 30:358-360.
	
10.	Stobetsky  V.I. (1988c).  On the nature of three cytogenetic
phenomena - delayed spiralization of metaphase chromosome portions ,
delayed disruption of telomeric links between chromosomes  and premature
centromere division of the X-chromosome . (Russ). Tsitologia .
30:1270-1272 .
	
11.	Stobetsky V.I., Mironova  L.L. (1988d). 5-Chlorodeoxyuridine
induces the formation of specific  dicentric chromosomes in cells with
micronuclei more effectively than does 
5-bromodeoxyuridine. (Russ). Tsitologia. . 30:1498-1500.
	
12.	Stobetsky  V.I. , Mironova  L.L. (1989). Telomeric fusion of
chromosomes in cells with micronuclei under  subsequent  incorporation
of halogenated analogs of thymidine into DNA.  (Russ). Tsitologia .
31:1242-1244.
	
13.	Stobetsky  V.I.  (1990).  Induction  of  specific  dicentric
chromosomes in Chinese hamster ovary cells  CHO-6 . (Russ).
Tsitologia.32:92-94.
	
14.	Stobetsky  V.I. (1991a). The induced telomeric chromosome fusion
under conditions of pulse action of hyper- or hypothermia. (Russ).
Tsitologia .33:116-118.
	
15.	Stobetsky V.I.  (1991b). Telomeric chromosome fusion in cells
with micronuclei in the conditions of combined action of hyper- or
hypothermia and halogenated analogs of thymidine . (Russ).  Bull. Exptl.
Biol. Med. No7. 72-73.
	
16.	Stobetsky  V.I., Chebotarev  A.N., Mironova  L.L. (1994).
Analysis of  chromosome combinations in dicentric at the induced
telomeric fusion. (Russ). Tsitologia. 36:60-63.

****************************************************************************

******
Vladimir I. Stobetsky
Senior Scientist
Research Institute of Pediatric Hematology
Laboratory of Cytogenetics and Molecular Biology
App. 116, 6 Novoorlovskaya St. Moscow, 119633, Russia
tel: +7 095 731 8932   
e-mail address: sto at aha.ru  






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