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positive signals. In normal human mammary epithelial cells (NHMECs), pericentrin
positive signals were scored for the total number of cells with visible centrosomes,
demonstrating a dose increase in pericentrin positive signals in treated cells.
Multipolar spindles and multiple centrosomal bodies were observed in
Zidovudine-exposed NHMEC and were identified by localization of Aurora
A-positive signals in NHMECs exposed cells. Western blot analysis of NHMEC
lysates confirmed an increase in protein expression of Aurora A in exposed cells.
Additionally, Zidovudine genotoxicity was documented by the presence of
large bodies still attached to the nucleus containing kinetochore positive signals.
These types of lesions known as nuclear buds were further studied and charac-
terized in detail by Dutra et al. (
2010
).
The most current treatment for HIV-1 is combination therapy, or HAART
(Highly Active Antiretroviral Therapy), which typically consists of at least two
NRTIs and a protease inhibitor. Zidovudine is frequently used in combination with
other NRTIs such as 2',3'-dideoxy-3'-thiacytidine (3TC) and 2
0
,3
0
-dideoxyinosine
(ddI). Centrosome amplification caused by 3TC, d4T, and ddI was carried out to
determine if it was a common phenomenon to NRTIs since these drugs are often
used together in combination. CHO cells and two NHMECs, a high and a low
incorporator of Zidovudine into DNA, were used by Yu et al. (
2009
). Hamster and
human cells exposed to any of the three NRTIs for 24 h exhibited amplification of
centrosomes and human cells also exhibited disruptions in tubulin distribution,
revealing that centrosome amplification as well as tubulin disruption is a common
phenomenon induced by several different NRTIs.
Increase of centrosome amplification induced by thymidine was no statistically
different than the controls for these strains indicating that an effect independent of
nucleotide pool imbalance was taking place.
Since not all centrosomes are equal in terms of their microtubule nucleation
activity, there could be multiple scenarios for amplified centrosomes (Ghadimi
et al.
2000
). Active centrosomes can be defined as those able to nucleate micro-
tubules. These centrosomes will go on to form spindle poles during mitosis. Some
aberrant centrosomes may be inactive and therefore unable to nucleate microtu-
bules and form spindle poles. To determine if extranumerary centrosomes induced
by NRTIs are active, microtubule nucleation of NRTI-treated cells was interrupted
with nocodazole, a disruptor of tubulin polymerization. Once nocodazole was
rinsed off, active centrosomes began to form microtubule asters. These experi-
ments revealed that multiple centrosomes induced by NRTIs kept the ability to
nucleate tubulin and form asters. These cells have the potential to form multipolar
spindles during mitosis that lead to missegregation of chromosomes and inevita-
bly, aneuploidy.
The origin of the new centrosomes has been a topic of interest for many
investigators. To understand if the new structures have their origin as a conse-
quence of overduplication due to cell cycle alterations or if they are just amplified
independent of the cell cycle, Davila et al. (
2009
) measured centrosomal ampli-
fication in human hTERT transformed MCF10A cells, by immunocytochemistry
with antibodies to centrosomal proteins pericentrin and Cep 170. Furthermore, the
