Although a common occurrence, the diagnosis of prostate cancer is fairly impaired by in sufficient data and unclear understanding of the disease pathology. The development of various in vitro models that help in identifying a common thread in prostate cancer and thereby help in early detection of the disease can go a long way in providing a more successful treatment to this type of cancer.
Preclinical studies have always focussed on understanding the morphology and cellular mechanisms of the tumor cells involved thus providing more light on the disease per se. The use of in vitro models to understand the cellular, biochemical and molecular aspects of prostate cancer has in turn enhanced the process of diagnosis and early cancer detection.
REVIEW OF LITERATURE:
While there have been a number of different diagnostic approaches to determine the onset and presence of prostate cancer, the need for early detection of prostate cancer goes a long way in successful treatment therapy of the disease. The presence of a large number of prostate cancer patient pools would normally ensure that the disease pathway would have more or less been clearly understood by now. However, that is not the case. Given the large amount of people suffering from this type of cancer, the molecular and cellular aspect of prostate cancer remains highly inconclusive. The problem being the low amount of conclusive and proven data on these cellular aspects that are actually available in order to determine as to what path the newer models of diagnostic approach can undertake.
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This review takes a look at the various in vitro models that have been used to mimic the cellular and molecular functioning of the prostate cancer tumor cells and how useful they have all been in providing an alternate or different methods of detecting prostate cancer at the earliest stage thereby making treatment therapy more successful and available to a broader spectrum of patients.
 Diagnostic in vitro models have focussed on the theory of gene mutation and chromosomal aberration and goes on to test the possibility of using the gene in question as a model to understand the gene fusion reaction at the chromosomal level that in turn causes genes to switch on or off and in turn cause prostate cancer. These studies involve the use of the androgen controlled gene TMPRSS2.
A number of the above cell lines that are marked for detection by various micro assay techniques are obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured. Frozen and non purified prostate cancer cells were obtained and used as in vitro models and the genetically altered gene was marked with biomarkers and induced into the models to study the effect in the DNA of the gene inside the cancer target cell.
The above study shows the use of either genetically altered causal entity or the target molecule entity. The overall perspective of using gene recombinant technology as in vitro model of diagnosis and early detection of prostate cancer becomes clear. There are still various models that focus on the other aspects of using different models for the in vitro mechanisms of understanding prostate cancer.
Delineation of TMPRSS2-ERG Splice Variants in Prostate Cancer using in vitro model that focuses on the gene TMPRSS2-ERG and the changes that it brings about in prostate tumor cells. The gene is a variant is specific to translation and is identifiable with a marker after cloning and c-DNA synthesising and sequencing. It shows that a specific therapeutic target is available to show presence of prostate cancer.
There are also research ideas and theories based on a different trial that theorised on cryosurgery in vitro models in human prostate cancer cell lines PC-3 and LNCaP and how it affects treatment options in cryosurgical cases of prostate cancer.
The process of cancer development consists of initiation, promotion and progression. Therefore, the need to develop in vitro models that actually mimic the cancer cells and in turn help in understanding the pathogenesis of cancer arose. The idea behind this was to evaluate the use of chemopreventive agents as tools in understanding the cellular differentiation and proliferation of cancer cells. The evaluation of chemopreventive retinoids using a in vitro model of prostate cancer cells provide a better understanding of the process of carcinogenesis. Retinoids are essential for normal epithelial differentiation and results from the above study could be used in understanding the disease thereby helping in early detection of cancer.
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The process of carcinogenesis is a very specific process of cell signalling and transduction in the various cell pathways that in turn need a large amount of target cells that can be studied in order to understand and provide a better view of the various minute changes that occur in a cell during the formation of cancer. Hence, the need to develop models that do not affect the cell growth and proliferation in any manner arises. This methodology was followed by one particular study that researches the possibility of using an intracellular signalling molecule in hepatocytes that both proliferate in plenty and provide an overview of all the biochemical pathways triggered by the ribozyme transgene molecule used as an in vitro agent.
However, the one major drawback of the genetic recombinant in vitro models are that they are purely one dimensional and need a single robust assay that determines the various gene segments; both for prostate cancer and other cancers. Therefore, the need to develop better in vitro models that took a 3-dimensional approach arose. One study focuses on developing 3D model of the human prostate cancer in vitro and also be able to stimulate and characterise it on the cellular and molecular levels. This approach provides a truly specific trial of approach that is not merely speculative. There are two main components in this study:
Development of 3D Culture of Human Prostate Cancer Cells under Perfusion.
In order to increase the size of the 3D cancer constructs, the study perfused the constructs with culture medium to increase the delivery of nutrients and removal of wastes. The perfusion did not alter the overall morphology of the LNCaP cells. Thus the study decided to use endothelial cells to shield the cancer cells from shear stress as is the case for in vivo systems.
Fabrication of tubular PGS to improve nutrient delivery and enhance waste removal without endangering the 3D in vitro cells.
The article focussed on using genetically altered gene structure in the retroviruses and then being induced into the cells they infect. This would provide a understanding of how the target cells interact with the retrovirus molecule and intra cellular signalling. Therefore, it provided a method of using the causative agent as a recombinant molecule and then understanding the process of prostate cancer. The project thus provided a view of how the retroviruses are trafficked into the cell and a surface view of the biochemical pathway of the disease.
Another study of hyaluronic acid based hydro gels as 3D matrices in in vitro study of chemotherapy agents of poorly adherent prostate tumor cells. Thereby, providing a three dimensional understanding of the methodology of the model. Again, the use of cell cultured prostate cells were used a in vitro model. Although developed in the late 1980â€™s, this model has remained the gold standard in anti cancer discovery till the present time.
The important aspects of all the above models are that they constantly focus on the gene recombinant and molecular aspect of the disease. This is a fool proof approach to any disease as it provides the investigators with a specific genetic map of the tumor cells and in turn help in understanding the disease at the cellular level. The various methodologies used by the in vitro models tell us as to how the tumor cells act to the specific genetic tweaking and how they react to those changes at the molecular level. In a way, they provide us with a third person perspective of how the cancer cells act. The development of a in vitro model that focuses on the cellular functions from the first person perspective with respect to the tumor cells has in turn added a lot more credibility to the early detection of prostate cancer and one such case serves as the case study for this review article.
 A New Cell Line Expressing A Novel Type Of TMPRSS2-ERG Gene Fusion Derived From Primary Tumors Of Familial Prostate Cancer Patient.
In this study, the tumor tissue (RC-123T) used for generating the RC-123T/E cell line with HPV-16 E6E7 genes was obtained from a 57 year old familial prostate cancer patient who had well differentiated adenocarcinoma. The cell line was characterized phenotypically and cytogenically. Dual colour fluorescence in situ hybridization (FISH) assay was used to test for ERG (3' 5') break-apart, and for translocation of TMPRSS2 and ERG and ETV1 [genetic variants], in both interphase nuclei as well as metaphases. The cells were also characterized for purported stem cell markers CD133, SOX2 and prostate cell markers by immunological history and RT-PCR.
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The study concludes that the RC-123T/E cell line and its primary tumor tissue possess the translocation of TMPRSS2/ERG to chromosome 7q which adds another class of gene arrangement in prostate cancer. In addition, this cell line expressed putative stem cell markers and TMPRSS2-ERG gene fusion. This model provides a novel tool to study the cellular and molecular mechanisms of TMPRSS2-ETS genes in prostate cancer.
The case study taken up in this review article is one of the most common type of gene recombinant method of in vitro model that focuses on using a recombinant gene of the ECR type and then using it in an in vitro prostate cancer cell to mimic the process of cellular and molecular cell signalling that in turn throws some possibilities in the diagnosing and treatment approach to the challenge that prostate cancer is. The most difficult challenge is to maintaining the steady proliferation and culturing of the tumor cells in the laboratory and then using them to trigger cell specific reaction in cells that are maintained in vitro.
The study is significant as it uses the tumor cells induced to proliferate into the new cell line that specifically expresses a particular HPV-16-E6E7 genes and the translocation of a specific TMPRSS2-ERG and ETV1 region on the tumor cells. These translocations happen during the metaphase and interphase of cell division.
This model successfully provides a means of understanding the molecular aspects and the very mechanism of cellular functions. Thereby it explains the cause of the mutation in the epithelial cells that in turn elicits a cell specific response and hence helps us get an idea of how the cells react to certain genetic tweaking.
Based on the different models of in vitro techniques, one can broadly classify the various models on four grounds:
[2,5,10]In vitro models that use TMPRSS-2 gene recombinant method and prostate tumor cells as in vitro cell lines.
[1,7]In vitro models based on the 3D method of building and stimulating cell signalling and molecular transduction thereby providing an understanding of biochemical cellular pathways.
[3,4,6,8,12]The in vitro methods that provide an alternate method of diagnosis of prostate cancer using prostate cells and chemotherapeutic isotopes that are measurable by specific assays and molecular techniques.
[9,11,13]In vitro models that focus on other cells, genes and variations of TMPRSS2 gene.
The most important highlights of the various in vitro models mentioned are:
Identification of a specific TMPRSS2 gene and targeting it to provide knowledge on the cellular mechanisms and molecular functions
Provide an accurate understanding of prostate cancer.
Help in detecting and early targeting of cancerous growth.
However, these models of exploration of the phenomenon that prostate cancer is, tell us how the cancer cell cultures react to the various external forces that act upon it and how it elicits a specific response from the cell to manifest itself and express itself as a chromosomal entity, the life of the gene molecule. They are only speculative on why the reacts and expresses itself the way it does to those external factors and forces.
The 3D in vitro models help in the understanding of the structural entity of the cell becomes much more clear as it involves the 3D development of cell lines of prostate cancer cells and stimulate them at artificial conditions to enable them to change and become sensitive to a cell signalling rhythm that in turn influences various cell transduction and ribosomal changes and they provide an over view of biochemical pathways. These studies tell us as to what happens at the DNA level of the cell and why the cell expresses itself to a particular response.
The use of chemotherapeutic isotopes in vitro, tell us the changes that induced target foreign particles have on the in vitro cells and how accurate the readings are by using cell and chemo isotope specific cellular and protein assays. They only mimic already assumed and expected response and thus are not broadly specific. However that may be, these models are very useful in providing an understanding of known phenomenon and hence provide an area of improvement and likely therapy and identification methods.
The fourth class on models go one step further and probes the effect of gene recombinant variations of the standard TMPRSS2 gene and its co molecules. Thus, these also provide a futuristic catharsis of the fairly predominant form of cancer.
The use of in vitro models as a diagnostic approach in prostate cancer definitely is of prime importance as these models simulate the natural conditions under which the cells operate and function. Thus, we can utilise these in vitro techniques to enable us to understand what changes occur inside the cells and what are the specific parameters that cause a normal cell to become cancerous.
The use of gene recombinant technology is an added advantage as they help us in understanding the specific target sites on the cell lines and thus enable a better and more rounded approach to decoding the process of prostate cancer.
Although, prostate cancer is a fairly predominant disease, the non availability of data, recorded information and conclusive evidence based result has made the early detection of this type of cancer rare and difficult. The use of gene recombinant technology seems promising in overcoming this difficulty. The use of specific gene targets and variants ensure that the exact area of interest of the disease can be monitored at all times thereby making it impossible to miss out even the slightest of detail. Also, the use of gene recombinant therapy as a diagnostic tool is more likely to succeed as they target specific areas of the tumor cells. The use of genes that are regarded as the molecular and functional unit of a cell as vehicles of recombinant technology that in turn influence various mechanisms in the DNA certainly provides a sense of uniqueness that can be compared to a lock and key model which is very specific.
However, as is always the case with newer approaches to a disease, more research and ideas have to be developed and conclusive and irrefutable evidence is needed before the use of such in vitro models can be essentially applied in day to day cases of diagnosis of prostate cancer.
The original case study of this review article however is very different in its approach as it provides a new cell line of in vitro models for the gene recombinant therapy of TMPRSS2 gene fusion and its impact on tumor cells. It therefore is truly a newer and definitely a novel way of approach to diagnosing prostate cancer as it also helps in understanding the cellular and molecular functioning of the cells. Even though the cell cultures are obtained from the same source as primary tumors, they express gene fusion of a specific TMPRSS2 gene and thus make the approach specific and accurate at the same time.
The cell line culture was obtained from epithelial cells of a 57 year old patient of prostate cancer. The cell line was named RC-123T/E and was characterised for putative stem cell markers that in turn ere measurable by various immunoglobulin and micro assay techniques. The response and expression of a androgen regulated prostate specific gene and the cytokine gene were recorded and was found to be quite significant.
The translocation of a specific gene segment in the RC-123T/E cell line was due to chromosome 7q which adds a different sequence of gene segment at the same chromosomal loci. Thus, the measurable quantity here is the intended chromosomal aberration at a specific gene locus to study a specific effect in the in vitro culture media. In addition to this, the study also concludes that the cell line recognised stem cell markers and fusion of TMPRSS2-ERG gene fusion.
Thus, it provides a molecular and cellular aspect of a highly complex and inconclusive type of cancer. The case study is unique in its method of approach as it deals with the development of a completely new cell line to be used as in vitro model in probing the cause and effect of various genetic factors on prostate cancer cells. The most evident outcome of this case study model is that, while the existing methods provide an understanding of how the cells act to various genetic recombinants and how the cells mutate to progressively develop into cancerous cells, the in vitro model suggested in the case study goes one step further and tells us as to why the cells react the way they do to these genetic changes. This is interesting as it provides us with a first person perspective from the point of view of the cancer cells. Thereby, it is almost as if the cancer cells themselves tell us as to why they act the way they do to the various changes and in turn progressively form the disease.
However, more such models are needed before their utility in the future diagnostic approaches to prostate cancer can be determined. The problem with prostate cancer being the surprisingly low amount of conclusive and authentic data on the disease considering how common a occurrence prostate cancer is. A large number of males in the age group of 45-70, and yet the amount of recorded information on the molecular and cellular pathways is limited.
Thus, it is a very nascent model of in vitro methodology and shows promise as the future standard of in vitro models.
However, more study and better improvements of this model has to be developed before a conclusive decision can be taken on the development new cell lines as in vitro models.
The need for good culture cell lines and proliferating cells make the use of regenerative hepatocytes as in vitro models also is another area that can be incorporated into the methodology of the case study so as to facilitate and support both, understanding and more cellular and biochemical simulation of the target cells and genes. These methods provide as a means of future catharsis in the diagnosis of prostate cancer.