LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL TUMOURS
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Date
2012
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
Therapeutic decisions are determined by proliferative behaviour of a tumour and
this is measured by determining the mean growth fractions and mean cell cycle time.
AgNORs (Argyrophilic Nucleolar Organising Regions) are only parameter available on
formalin fixed tissues to measure proliferation fraction of tumours. AgNORs have the
advantage that their numbers are increased only in actively and fastly dividing cells.
Most of the sites of the NOR proteins reactive with silver are their Zinc binding motifs.
These motifs remain bound with zinc particularly in their active states. The chemical
reaction and mechanism involved in impregnation of silver at these sites of NORs are
such that they can be greatly influenced by presence or absence of zinc in these zinc
motifs. The process of cell division is initiated by replication of DNA along with
Abstract……… Page 2
increased rate of transcription. Both these processes are initiated and mediated by a
number of Zinc binding replication and transcription proteins.
These zinc binding proteins are highly dynamic and transportable to different
nuclear compartments engaged in transcription or replication of genes. Thus we
hypothesised that presence of zinc bound proteins at different levels and their
distribution in different compartments of nuclei as well as nucleoli in different
metabolic and proliferative states of the cells have definite impact on AgNOR number
and clusters which need to be explored. This warranted localization of zinc binding
sites in nuclei and their further co-localization with silver by the method of AgNOR in
order to throw more insight on mechanism involved in formation of AgNORs and
variation in their number, size, shape and clusters in varying metabolic and
proliferating states of the cell as well as in different phases of cell cycle. Keeping these
facts in mind the present study aimed at development and standardization of some new
protocol for co-localization of zinc and silver binding sites in nuclei and to devise a
system of morphologic pattern of these granules to identify and count the cells in
different phases of cell cycle, which can evolve as a more perfect marker of speed of
proliferation.
For localization of zinc, three methods were tested which were zinc
impregnation, zinc precipitation and zinc-cysteine complex formation methods.
Attempt to localize zinc by impregnation and precipitation methods caused nonspecific
deposits in nuclei as well as in cytoplasm. This made intranuclear specific
deposition of zinc obscured.
The problem of nonspecific deposition of zinc was removed by treating the
sections with solution of ionic Zinc, Na2SO3, acetic acid which caused deposition of fine
amorphous to crystalline, well discernible and discrete granules or dots mostly
restricted in nuclei. These granules were taken as specific since this staining reaction
aimed at formation of insoluble complex of Cysteine and Cysteine bound zinc.
Abstract……… Page 3
Moreover, these granules or dots were further histochemically confirmed for presence
of zinc with Dithizone method. This reaction was based on previously known reaction
for Cu during which there is formation of insoluble Cu-Cysteine-Acetic Acid complex
when solution of Cu and Cysteine were treated with solution of Na2SO3 and acetic acid.
Keeping this reaction in mind it was hypothesised that cysteine bound with zinc may
lead to formation of Zinc-Cysteine-Acetic acid complex when a section pre-exposed
with ionic zinc is treated with a solution of acetic acid and Na2SO3 which may appear as
visible dot or granule.
Thus this method could localize only those zinc binding sites which were bound
with cysteine such as polymerase, and other transcription factors. In consequence of this
test result, it was adopted for intranuclear co-localization of zinc and silver binding sites
in nuclei.
When dots due to AgNOR were compared with co-localized Zn-AgNOR dots, in
these co-localized dots, there was presence of markedly more number of fine visible
discrete dots dispersed throughout the nuclei along with few large Zn-AgNOR dots
closely resembling to AgNOR dots formed due to AgNOR staining alone. A consistent
appearance of these small fine AgNOR dots along with large Zn-AgNOR dots indicated
sites of transcription of non-NOR genes as well as replication sites of DNA. Such sites
might have failed to be visualized by AgNOR staining due to very poor silver reaction
there and treatment of zinc on these sites made them strongly reactive and clearly
visible. Thus increase in their number and density indicated enhanced transcriptional or
replicational activities in cells.
When co-localized Zn-AgNOR dots were further stained with Dithizone, the fine
granules were found to be more distinct because of coloured reaction as well as due to
mounting and refractive index used in this procedure. This staining reaction due to
combination of Zn-AgNOR-Dithizone staining could be able to indicate whether Zn-
AgNOR dots are rich in zinc or silver, red dots (Dithizone stained Zinc) rich in zinc
Abstract……… Page 4
indicated active sites of transcription or replication whereas black or brown dots might
be representing presence of silver binding transcriptional or replicational proteins.
A thorough and critical study of morphologic pattern of Zn-AgNOR-Dithizone,
Zn-AgNOR dots as well as AgNOR dots revealed variable pattern in different cells.
Nuclear proteins are highly dynamic and such proteins from nucleolar compartment
are migrated to different replicating and transcribing sites in nuclei. Moreover, during
process of replication almost all the clustered genes are unwound and loosened. These
might be responsible for dispersion of nucleoli in S-phase and formation of small dots
dispersed throughout the nuclei.
Due to doubling of DNA and aggregation of transcription proteins like
polymerases and upstream binding factors (UBF) with actively transcribing genes to be
packaged in chromosomes; dots in G2-phase were comparatively larger than that found
in S-phase.
In M-phase Zn-AgNOR dots were arranged in forms of mitotic figures showing
clumps or aggregation of granules.
G1 phase was characterized by presence of one or few nucleoli with variation in
their degree of organisation represented by varying size of large NORs with fine well
dispersed in Zn-AgNOR dots in nuclei.
In highly proliferative cells there may be more number of irregular compound
dots along with focal presence of features of S or G2 phases indicative of fast replicative
and transcriptive activity in the cells. These cells in this study were graded as irregular
or aberrant cells.
In this way this system of morphologic pattern of Zn-AgNOR dots enabled us to
identify and count the relative proportion of cells in different phases of cell cycle.
Counting of cells by this system revealed a major relative proportion of cells in each
phase of cell cycle and consequently it also indicated the timing of each phase and thus
it can give an estimation of cell cycle velocity and proliferating fraction of the tumour.
Abstract……… Page 5
The higher count in S or G2 phase with lower count in M-phase indicated that the
majority of cells are in synthetic phase (S-phase) with slow rate of proliferation.
Likewise, higher cell count in S-phase as well as in M-phase both indicated higher rate
of proliferation and represent a fast or newly developing tumour.
This above mentioned system of phases of cell cycle was further applied to
different tumours of mammary gland origin (tubular, solid, anaplastic, mucinous,
myoepithelioma and osteosarcoma) and seminoma to assess their proliferative
behaviour. In order to see the accuracy of this assessment it was further tested for
positive correlation with count of mitotic figures (H&E staining) as well as histological
grading of these tumours.
A general trend of variation in mean count of cells in S/G2+M (taken as
proliferation fraction) in almost all tumours was consistently found to be either
significantly or non-significantly higher in Grade III followed by Grade II and lowest in
Grade I lesions of their tumours. This clearly indicated highly proliferative population
of neoplastic cells in tumour of Grade III followed by Grade II and thereafter in Grade I.
These results clearly showed an association between assessment of proliferation made
by our system of cell count and histological grading of tumour suggesting that both are
positively correlated. In majority of cases assessment of histological grading of tumour
was also found positively correlated with cell count in different phases of cell cycle.
Zinc-AgNOR-Dithizone staining showed best result while identifying,
differentiating and counting the number of cells in different phases of cell cycle
followed by Zinc-AgNOR co-localization while AgNOR could reveal these feature in
case of one or two tumours.
In this way, this system could stand in association with histopathological grading
of tumours as well as their morphological behaviour. It was also able to explain number
of aspects pertaining to developmental and biological behaviour of different tumours
thus it could be concluded that this system is able to identify cells in different phases of
Abstract……… Page 6
cell cycle on the basis of pattern of Zn-AgNOR dots and can help in assessing
proliferative fraction as well as proliferative behaviour of tumours which has got
immense importance in determining prognostic and therapeutic decisions on them. It
could be concluded that use of histochemical staining of sections using Zn-AgNORDithizone
or Zinc-AgNOR for identification and counting of number of cells in different
phases of cell cycle has potency to evolve as important histochemical technique to
assess the proliferative behaviour of tumours which can be helpful in taking therapeutic
and prognostic decision for them.
Description
LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL TUMOURS
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