USE OF ACRIDINE ORANGE STAINING FOR SIMULTANEOUS ASSESSMENT OF PROLIFERATIVE AND APOPTOTIC ACTIVITY IN N-NITROSO-N-METHYLUREA INDUCED TUMOURS IN RATS
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Date
2015
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
Biological activity of a tumour is determined by a balance between proliferative and
apoptotic activity of its neoplastic cells. Histopathological typing and grading has remained a
gold standard to assess proliferative aggressiveness of a tuomour which relies mostly on
anaplastic and apoptotic changes shown by neoplastic cells.But many times these changes do not
reveal true proliferative behavior of a tumour. These problems motivated the augmentation of
histopathological methods to improve the accuracy of assessment of proliferative and apoptotic
activity of tumours.
Proliferative behavior of a tumour is determined by mean growth fraction and mean cycle
time of their neoplastic cells. Growth fraction of tumour in a tissue section is assessed by
measurement of proliferation associated antigens like PCNA, Ki-67 etc. But no technique is
Abstract……… Page 2
available to see the speed of proliferation of neoplastic cells particularly in terms of mean cycle
time instantaneously in tissue section of tumours. This highlights the importance of identification
and quantification of cells in different phases of cell cycle to throw more insight on proliferative
speed and fraction of neoplastic cells.
Different protocols of flow cytometry for identification and quantification of neoplastic
cells in different phases of cell cycle in isolated cell system are available using acridine orange
(AO) staining. These methods can not reveal complete picture of location, migration,
differentiation and relation of dividing cells with other cells in their true histological architecture.
So in this study an attempt was made to establish a protocol to identify, localize and quantify
such cells in microscopic sections of different NMU induced tumours in Wistar rats.
Moreover, AgNORs are the only parameter used to assess proliferative fraction as well as
proliferative speed of a tumour on a tissue section indirectly. It has the advantage that their
numbers are increased only in actively and fastly dividing cells. The major problem in
acceptance of this method as an indirect marker of proliferative speed in terms of mean cycle
time is deposition of innumerable fine dots in nuclei in background of large AgNOR dots which
has been greatly variable from cell to cell depending upon their metabolic and proliferative
status, most possibly in different phases of cycle. Hence, critical analysis of variation in pattern
of deposition of such small dots in nuclei has scope to evolve as a system for identifying cells in
different phases of cycle. This motivated to evolve one more new system of identifying and
localizing dividing cells in different phases of cell cycle on the basis of variation in pattern of
deposition of such Non-AgNOR dots in nuclei in histological sections after AgNOR staining.
The procedure for optimally demonstrating highest intensity of fluorescence of DNA and
RNA along with cellular characteristics for necrotic, apoptotic and neoplastic cells passing
through different phases of cell cycle as described and accepted by the method of flow cytometry
was accepted and being described as follows.
For acridine orange staining of formalin fixed paraffinzed tissue sections of tumours were
treated with 0.025% AO in 1 % acetic acid for one minute and then were washed in M/15
Phosphate Buffer (pH 6.0) for few second and sections were mounted in a drop of phosphate
buffer and used for examination under fluorescent microscope to identify and count different
types of cells.
Abstract……… Page 3
The characteristic feature accepted for identification of cells in different phases of cell
cycle was based on variation in the colour of fluorescence given by nucleus and cytoplasm of the
cell. Dull green fluorescing nuclei and cytoplasm was characterized as cell in G1 phase whereas
dull green nuclei with red cytoplasm was characterized as late G1 phase of cell cycle. The cells
with bright green to white yellow nuclei with orange cytoplasm were identified as S phase. S/G2
phase of the cell cycle was characterized by bright green to yellow-white nuclei with red
fluorescing cytoplasm. The cells with bright green nuclei with mild or no red cytoplasm were
identified as M phase of the cell cycle. Apoptotic cells were characterized by condensed orange
to red nuclei with red cytoplasm whereas cells with homogeneously fragmented red fluorescing
nuclei were identified as necrotic cell.
These characteristic feature was accepted for identification of cells by AO staining was
highly comparable with those well accepted for flow cytometry taken as standard for
identification of such cells in this study. However identification of cells in histological section by
AO staining have advantage over flow cytometry that it gave complete picture of apoptotic
activity of tumours in terms of their number, location, migration, differentiation and architectural
arrangements of dividing cells.
One more new system to identify and localize proliferating cells in histological sections
was also evolved using AgNOR staining after a critical perusal of variation in pattern of
formation and distribution of non- AgNORs along with their nature of persistent reproducibility
and ability to be dissolved in sodium thiosulphate solution in AgNOR stained sections of
different tumours. The characteristic features incorporated in this system were based on
formation and distribution pattern of AgNOR dots and non-AgNOR dots in the nucleus. G1
phase was characterized as cells having large AgNOR dots with few small or medium sized non-
AgNOR dots. The Cells with innumerable very fine and small non-AgNOR dots with or without
large AgNOR dots inside nucleus was in S phase. G2 phase was characterized as cells having
small to medium sized innumerable non-AgNOR dots with brown backgrounds whereas cells
with black dense irregular hairy nucleus was considered to be in M phase. The cells in S, G2, and
M phases of cell cycle were collectively considered to be in proliferative phase.
The accuracy of both these systems to identify, localize and quantify neoplastic cells in
different phases of cell cycle was further verified by seeing a correlation between counts of such
Abstract……… Page 4
cells in histologic sections of different types of NMU induced tumours after AgNOR as well as
AO staining.
Keeping these facts in mind, both these systems were further validated by seeing a
correlation between counts of cells in different phases of cycle after AgNOR staining and
percentage count of quiescent, proliferative and apoptotic cells after AO staining with
pathomorphological aggressiveness of different types of NMU induced tumours in wistar rats
assessed in terms of tumour volume, multinodularity, total cell count and their impact on survival
period.
Out of 60 female virgin Wistar rats of about 35 days of age were intoxicated with NMU
at the dose rate of 50mg/kg body weight intraperitoneally at 50th, 57th, 64th and 71st days of their
age; In order to obtain complete pathomorphological behavior of NMU induced tumour, all the
experimental animals were allowed to survive till they died either due to growth of tumour or
due to NMU toxicity which prolonged for one year. A total of 44 tumours could be induced in 32
tumour bearing rats. Tumour incidence was found to be 53.33%. The average number of tumours
per animal (tumour frequency) was found to be 1.33. The average survival period was 276 days.
The mean value of nodularity was found to be 3.25 among the NMU induced tumours. The
average volume of the tumour was found to be 26.18 cm3.
These induced tumours were further classified into five histologycally distinct types
which were mucinous adenocarcinoma (10), tubular adenocarcinoma (7), stromal cell sarcoma
(8), mammary ductular adenocarcinoma (12) and gastric squamous cell carcinoma (7) which
revealed almost their characteristic histopathological features.
Count of cells in different phases of cell cycle after AO and AgNOR staining in
histologic sections of almost all these different types of NMU induced tumours were found to be
highly and positively correlated and thus comparable with one another. It supported the validity
of both these systems to identify cells in different phases of cell cycle. Sensitivity of both these
systems were further verified indirectly by correlating the count of cells in different phase of cell
cycle with that of proliferative aggressiveness of all these different types of NMU induced
tumours as assessed by their volume, total number of cells, multinodularity, grading and survival
period which were mostly found to be positively and significantly correlated favouring the
ability of this system to identify cells in different phases of cycle.
Abstract……… Page 5
A critical perusal of variation in number of cells in different phases of cycle and thereby
relative timing taken by neoplastic cells in different phases failed to show a general trend of
variation in different histologic types of tumours. Rather, number of cells in different phases of
cycle was greatly variable in individual tumour to tumour. It warrants the count of such cells in
individual tumour, the critical analysis of which may throw more insight on their proliferative
behavior. Higher count of cells in S-phase, G2 phase as well as in M-phase indicated presence of
more actively proliferative population and actively growing tumour. Such findings can also
suggest that such tumours will give more response with chemotherapy since these agents act
mostly on replicating cells
A simultaneous count of cells undergoing apoptosis along with count of cells in different
phases of cycle will help not only in assessing proliferative behavior of tumour rather it may be
of great help in assessing sensitivity or resistance of tumour to a particular drug because different
drug acts through different mechanism, which involved inhibition of cells in different phases of
cycle or by inducing apoptosis. Mutational changes in these loci may be responsible for a
refractory of a tumour to different therapeutic agents. These protocols have a potential to evolve
as a test to help in selection of most appropriate chemotherapeutic agent against a particular
tumour.
Thus it could be concluded that both these systems are helpful in identifying and counting
of cells in different phases of cell cycle along with number of cells in apoptosis and can help in
throwing more insight on proliferative fraction as well as proliferative speed of a tumour and
relative timings in different phases of their cell cycle. Such assessment has got immense
importance in prognostic, diagnostic and therapeutic decisions on tumour. However, both these
systems of counting cells in different phases of cycle or undergoing apoptosis have still many
scopes for improvements as well as exploration to make it more accurate reliable and usable in
other areas of tumour and cell biology.
Description
USE OF ACRIDINE ORANGE STAINING FOR SIMULTANEOUS ASSESSMENT OF PROLIFERATIVE AND APOPTOTIC ACTIVITY IN N-NITROSO-N-METHYLUREA INDUCED TUMOURS IN RATS
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