LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING MESENCHYMAL TUMOURS
Loading...
Date
2012
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
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 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 AgNOR 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 alongwith 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 compartment 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 method (hypothesized on basis of principles previously used for localization of argyrophilic proteins in nuclei) at their binding sites using citrate buffer, hydroquinone and citric acid as reducing agent as well as zinc precipitation method (based on principle of chromate precipitation technique used previously for demonstration of fine histologic structures like synapses of axons and dendrites) using thioglycolloic acid, sodium sulphide 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 AgNOR methods to co-localize silver on these fine particles improved their visibility seen as large round, black, non specific 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, sodium sulphite, 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 mechanism of previously known reaction during which there is formation of rich insoluble copper cysteine complex compound when solution of copper sulphate and cysteine were treated with solution of sodium sulphite (Ahmed et al., 2011). Keeping this reaction in mind, it was hypothesized that cysteine bound with zinc may also lead to formation of zinc-cysteine-acetic acid complex when a section pre-exposed with solution of ionic zinc is treated with a solution of acetic acid and sodium sulphite and may appear as dots or granules.
The size of granules were further enhanced by treating such sections with AgNOR, which resulted in increased number of fine granules in nucleoplasm along with more distinct presence of large AgNOR dots.
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.
In consequence of tests applied, most favourable results were adopted for intranuclear co-localization of Zn and silver binding sites in nuclei. Three methods of staining were proposed on the basis of these tests i.e. Zn-AgNOR-Dithizone, Zn-AgNOR and AgNOR alone.
When dots due to AgNOR were compared with co-localized Zn-AgNOR dots, these co-localized dots presented 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 transcription sites 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. But initial treatment with zinc made these sites strongly reactive to silver and clearly visible in case of Zn-AgNOR staining. Thus, increase in number and density of fine Zn-AgNOR dots indicated enhanced transcriptional or replicational activities in cells. Also, co-localized large Zn-AgNOR dots presented an important feature of cluster of fine, discrete and distinct dots within them in case of controlled stained sections; suggestive of multiple transcriptional sites.
Further Zn-AgNOR-Dithizone staining caused co-localized fine Zn-AgNOR dots to be more distinct because of coloured reaction given by dithizone as well as due to mounting media and refractive index used in this procedure. Some dots were red while many others were black or brown.It should to be noted that zinc gives red staining with dithizone while silver gives black. Such coloured 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 rich in zinc indicated active sites of transcription or replication whereas black or brown dots might be representing presence of silver binding transcriptional or replicational proteins.
Though counting and assessment of Zn-AgNOR dots in light of coloured reaction could not be included in this study. However, further improvement of this technique regarding this aspect can enable us to recognize active transcriptional and replicational sites in nuclei as well as to differentiate between active and inactive NORs.
Zn-AgNOR dots not only clearly and distinctly demonstrated even weakly reactive NORs, but also protected large dots from overstaining and strongly reactive NORs from confluence.
Varying morphological and distribution pattern of Zn-AgNOR dots as well as AgNOR dots in nuclei was thoroughly studied and cells in different phases of cell cycle were recognized. Cells presenting fine, innumerable dots distributed throughout nuclei without any large NOR dots resembling nucleoli were considered to be in S phase of replication.
Though transition between S phase and G2 phase has been difficult to delineate. But critical observation revealed that number of small fine dots in S and G2 phase remained almost constant. There was variation in size of dots, which were found to be larger in G2 phase.
In M phase, zinc and silver binding proteins packaged in chromosomes might be responsible for arrangement of Zn-AgNOR dots in form of mitotic figures showing clumps or aggregation of granules.
G1 phase was characterized by 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 indicated early or immature phase of G1 while their decreased number indicated towards maturity in G1 phase.
Some aberrant or irregular cells were having more number of irregular compound dots along with focal presence of S or G2 phase indicating high and independent replicative and transcriptive activity in the cells.
Such system of morphologic pattern of Zn-AgNOR dots enabled us to identify and count the 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; consequently indicating the timing of each phase along with estimation of cell cycle velocity and proliferating fraction of tumours.
Counting of number of cells in different phases of cell cycle in serial sections using Zn-AgNOR-Dithizone, Zn-AgNOR and AgNOR stains separately; along with counting of mitotic figure and grading of tumours in respective areas of other serial sections stained with H & E was done to assess the proliferative behaviour of mesenchymal tumours like venereal sarcoma, osteosarcoma, mammary osteosarcoma, mixed interstitial seminoma and synovioma.
The mean count of cells in S, G2 as well as S+G2+M taken as proliferative fraction of tumour along with mitotic figure were significantly higher in grade III followed by grade II and grade I in most of the tumour, suggesting their impact on morphological behaviour of the tumour.
The higher count of S+G2 phase (46%) and lower count in M phase (0.7%) in venereal sarcoma indicated that the major part of the cells were in synthetic or replicative phase but the timing of S phase was relatively longer and the rate of proliferation of cells were comparatively slow.
In osteosarcoma of lower grades, S+G2 phase (84%) was longer having low proliferating activity while in case of higher grades S/G2phase (25%) was shorter resulting into increased proliferative speed and behaviour of the tumour. Whereas mammary osteosarcoma revealed comparatively lower S+G2 count (grade 1 : 20%; grade 2 : 43%) with almost equal number of cells in M phase (grade 1 : 9%; grade 2 : 12%) in both grades of tumour, indicating small but highly proliferating pool of neoplastic cells. In higher grades of osteosarcoma, intracytoplasmic Zn-AgNOR dots were found in Zn-AgNOR staining or AgNOR alone.
Mixed interstitial seminoma during this study revealed comparatively lower S+G2 cells (11%) almost equal count of cells in M phase (9%), which indicated small but highly proliferative pool of neoplastic cells. Grossly tumour was of large size but histopathologically there was no marked degree of anaplasia. This small but highly proliferative pool might be responsible for its large size, though it failed to show marked anaplastic change.
In synovioma of lower grade, high number of cells contained more than five large, irregular compound dots (89%) in areas showing lower grade of tumour while higher count of cells in S+G2+M phases ( 98%) in areas of higher grades of lesions. Both these features clearly indicated this tumour to be highly and fastly proliferating.
In most of the mesenchymal tumours mean cell counts were found to be positively correlated with tumour grading as well as count of mitotic figure suggesting accuracy of these count for different phases of cell division . 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 lesions 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.
Qualitatively, co-localization of zinc and AgNORs with dithizone staining was found to give best result followed by Zn-AgNOR staining as compared to AgNOR alone in identifying and counting of cells in different phases of cell cycle. The superiority of Zn-AgNOR-Dithizone in assessing the proliferative aggressiveness of tumour was established on the basis of its ability to count maximum number of cells in S, G2 or M phases of cell division in correlation with histologic grading and mitotic figure count of that tumour.
Thus, it could be concluded that use of histochemical staining of sections using Zn-AgNOR-Dithizone or Zn-AgNOR for identification and counting of number of cells in different phases of cell cycle has potency to evolve as an important histochemical technique in assessing proliferative fraction as well as prolferative behaviour of tumour which have got immense importance in determining prognostic and therapeutic decisions on them. Moreover, these staining protocols and system of counting of cells has still many scopes for improvement as well as exploitation to make it more accurate, reliable and usable in other areas of tumour and cell biology.
Description
LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING MESENCHYMAL TUMOURS
Keywords
null