LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS
| dc.contributor.advisor | Singh, K.K. | |
| dc.contributor.author | Soren, Punam | |
| dc.date.accessioned | 2017-06-22T10:46:39Z | |
| dc.date.available | 2017-06-22T10:46:39Z | |
| dc.date.issued | 2012 | |
| dc.description | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS | en_US |
| dc.description.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 . | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810022854 | |
| dc.keywords | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS | en_US |
| dc.language.iso | en_US | en_US |
| dc.pages | 157 | en_US |
| dc.publisher | Birsa Agricultural University, Kanke, Ranchi, Jharkhand | en_US |
| dc.sub | Veterinary Pathology | en_US |
| dc.subject | null | en_US |
| dc.theme | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS | en_US |
| dc.these.type | M.V.Sc. | en_US |
| dc.title | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING HAEMOPOIETIC TUMOURS | en_US |
| dc.type | Thesis | en_US |