LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC 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 Ag-NORs are only parameter available on formalin fixed
tissues to measure proliferation speed of tumours. They have the advantage that
their numbers are increased only in actively and fastly dividing cells. The major
problem in using Ag-NOR counts as marker of proliferative speed are great
variation in count, size, shape and cluster of these NORs as well staining
intensity in different metabolic and proliferative states of the cell.
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 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 Ag-NOR number and clusters which need to be explored .This
warranted localization of zinc binding sites in nuclei and their further colocalization
with silver by the method of Ag-NOR in order to throw more insight
on mechanism involved in formation of Ag-NORs 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 protocols for colocalization
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, four methods were tested which were zinc
impregnation, zinc precipitation and zinc-cysteine complex and Zinc fixation in
tissue smear.
Attempt to localize zinc by impregnation method at their binding
sites using citrate buffer, hydroquinone and citric acid as reducing agent as well
as zinc precipitation method using Thioglycolic acid DTT ,sodium sulphide
(Na2S) as zinc precipitants caused deposition of very fine ,indiscrete and
nonspecific deposits when examined either by light microscope or phase contrast
microscope.
Further use of Timm’s or Ag-NOR methods to co-localize silver on these
fine particles improved their visibility but were seen as large round, black,
nonspecific deposits in nuclei as well as cytoplasm making intranuclear specific
deposition of zinc obscured .
The problem of nonspecific deposition of zinc was removed by
treating the section with solution of ionic zinc with Na2SO3 and acetic acid which
caused deposition of fine amorphous to crystalline, well discernible and discrete
granules or dots mostly restricted in nuclei. 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
dots or granules.
These granules were taken as specific since this staining aimed at
formation of insoluble complex of cysteine and cysteine bound zinc. Moreover,
these granules or dots were further histochemically conformed for presence of
Zinc with Dithizone method.
Thus this method could localize only those zinc binding sites which
were bound with cysteine such as polymerase, and other transcription factors.
Moreover, this solution could also remove zinc which were not bound with
cysteine and were deposited in nonspecific way since acetic acid in this solution
removes such zinc.
On the basis of these findings two protocols were developed for
co-localization Zinc and Silver in nuclei which are 1)Zn-AgNOR –Dithizone. 2)
Zn-AgNOR method. Another method developed during this study for colocalization
of Zinc by zinc fixative in tissue during preservation. It was based
on fact that, Most of the site of NOR proteins are reactive with silver and their Zn
-binding motifs that is Zn finger. These Zn finger remain bound with Zn
particularly in their active state. Impregnations of silver in these sites are such
that they can be greatly influenced by presence or absence of Zn in these fingers.
The proteins that bind Zn are easily denatured in water or any other liquid
containing fixative with loss of Zn ions from their fingers. It may alter the pattern
of Ag-NOR reaction because Zn bound and Zn free motifs had different kinetic
features for this reaction and may fill to revealed real pattern of NOR formation.
Moreover, such alteration might be responsible for lack of
reproducibility as well as variation in pattern of Ag-NOR formed by existing
methods of their staining. Keeping these facts in mind, after trial and
standardization of number of Zn precipitant, we formulated new composition of
formalin fixative which could precipitate Zn at their original motifs during the
process of fixation. After fixation tissue were washed, processed and embedded
in paraffin. Three to four micron thickness were cut and stained with AgNOR
staining as described earlier for co-localization of Zn-AgNOR dots.
Zn fixed AgNOR staining showed best result while identifying
differentiating and counting the number of cells in different phases of cell cycle
followed by Zinc-AgNOR-Dithizone, zinc-AgNOR co-localization while AgNOR
could reveal these feature in case of one or two tumours.
When dots due to AgNOR were compared with co-localized Zn-
AgNOR dots, there were 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
consistence 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. Another important feature of co-localized Zn-AgNOR dots was that even
in large dots resembling in AgNOR dots, there were presence of cluster of fine
discrete and distinct dots within them in case of controlled stained sections
suggesting them to be of multiple transcriptional sites.
Qualitative assessment of morphological characteristics of zinc
binding and argyrophilic sites appearing as dots were assessed by all the
4methods described above was made and a system was devised on the basis of
these characteristic patterns to identify the different stages of cell cycle.
i) S-phase- replicating cells in s-phase were identified on the basis of presence of
small sized innumerable dots in nuclei with no large dots appearing as nucleoli
ii)G2- phase –cells in G2 phase were identified by their pale-brown–orange
condensed background with innumerable small to medium sized dots having no
large dots appearing as nucleoli comparatively lower in number than in S-phase
iii)M-phase – The cells in mitotic phase were showing presence of dots and
granules regularly or irregularly aggregated in the form of mitotic clot or mitotic
hairs or mitotic figures were classified as cells in M-phase.
iv)G1-phase – The cells in G1- phase showed presence of single or few dots
within the nucleus presenting nucleolus along with presence of numerable small
dots in nucleoplasm .Presence of more number of such large dots indicate early
or immature phase of G1 while their decreased number indicated towards
maturity in G1 phase.
V) In highly proliferative cells there were more number of irregular compound
dots along with focal presence of features of S or G2 phase indicative of high and
fast replicative and transcriptive activity in the cells taking within short cycle
time of G1.These cells in this study were graded as irregular cell or fast G1.
The literature about the use of system of classification of
lymphomatous lesions in birds for their typing their grading as used in
mammals appears to be very scarce. Any system of typing and grading aims at
evolution of proliferative behaviour, prediction of prognosis and assessment of
survival rate. Hence attempt for typing and grading of lymphomatous lesions of
birds using these systems may give important feedback for improvement of these
systems of classification in their purpose. So in this study we have attempted to
classify lymphomas of birds in different organs on basis of WHO system of
classification for lymphomas and to assess the proliferative behaviour of these
tumours with our system of identification and counting of cells in different
phases cell cycle.
On the basis of WHO system, the lymphomatous tumour found in birds
were classified as lymphoblastic and lymphocytic lymphoma (Grade1, Grade2
and Grade 3).
A general trend of variation in mean count of cells in S, G2 and M
(proliferating fraction) of tumour in almost all the tumour was consistently
found to be either significantly higher in grade 3 followed by grade 2 and lowest
in grade 1 of their tumour. This clearly indicated highly proliferative population
of neoplastic cells in tumours of grade 3 followed by grade 2 and thereafter in
grade 1. 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.
Further mitotic figure counts were found to be positively correlated
with number of cells in different phases of cycle(S, G2 & M) in different grades of
tumour validing the accuracy of identification of cells in different phases. It is so
because increase in number of mitotic figures will associated with atleast one or
more of the M,S,G2 or M+S+G2.
Zinc fixed AgNOR stains was found to be superior than others because count of
cells in different phases of cycle was consistently more(S,M,G2). It also enabled to
differentiate between S & G2 phases in lymphomatous lesions.
It was concluded that use of histochemical staining of sections using Zn
fixed AgNOR or Zn-AgNOR-Dithizone or Zinc-AgNOR or AgNOR alone 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 HAEMOPOIETIC TUMOURS
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