LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL TUMOURS
| dc.contributor.advisor | Singh, K.K. | |
| dc.contributor.author | Verma, Ashok | |
| dc.date.accessioned | 2017-06-22T11:07:19Z | |
| dc.date.available | 2017-06-22T11:07:19Z | |
| dc.date.issued | 2012 | |
| dc.description | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL 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. 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. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810022868 | |
| dc.keywords | LOCALIZATION OF ZINC BINDING SITES IN NUCLEI TO BE USED AS PROLIFERATION MARKER FOR NATURALLY OCCURRING EPITHELIAL TUMOURS | en_US |
| dc.language.iso | en_US | en_US |
| dc.pages | 131 | 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 EPITHELIAL 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 EPITHELIAL TUMOURS | en_US |
| dc.type | Thesis | en_US |