DISCUSIÓN DE RESULTADOS 3.1 Análisis descriptivo de las variables de estudio

The cell line immortalised from the cells from the TUR chips from the deep region of the prostate produced anchorage independent colonies in agar without radiation manipulation after passage 9, (Figure 3.1a, Figure 3.1b). This was observed while undertaking the control experiments when irradiating P21d and growing the irradiated cells in agar to see if they had transformed. Frozen vials of P21d from passage 5 were grown up and the P21d cell line always produced anchorage independent colonies with increasing passage number after passage 9. As the early P21d was re-grown a few times and each time anchorage independent colonies were seen with increasing passage number it was deduced that the cells contained a latent cancer clone present in the population that had been immortalised with cdk4 and hTERT. This is more probable than spontaneous transformation for two reasons. One, spontaneous transformation of human cells in culture is an extremely rare event (Morales, et al., 2003; Herbert, et al., 2001) and the P21s cell line never produced anchorage independent colonies and it was immortalised with over expression of the same genes. Spontaneous transformation requires mutations in several genes, such as p16, p53, and pRb, and their cellular pathways that are involved in cellular senescence (Herbert, et al., 2001).

Two, prostate carcinomas arise from the deep region of the prostate so it is very probable that there was a latent clone present, (Long, et al., 2005). It has been estimated that 80% of males over the age of 80 harbour microscopic, undiagnosed, latent foci of prostate cancer (Taichman, et al., 2007). Most of these foci never develop into clinically detectable cancer which is consistent with the frequent finding of prostate cancer during autopsies of asymptomatic men in whom the condition was never diagnosed (Gelmenn, 2008; Stemmermann, et al., 1992).

Generally, transformation of cells in culture is defined by the ability to grow in an anchorage independent fashion, the ability to grow in the absence of serum, the ability to grow in soft agar, and by the development of tumours in nude mice (Morales, et al., 2003). Many cell lines immortalised with hTERT do not exhibit changes typically associated with malignant

transformation including being able to grow colonies in agar. This has been shown for human fibroblasts (Morales, et al., 1999), human oesophageal squamous epithelia cell lines (Morales, et al., 2003), and human bronchial epithelial cell lines, (Ramirez, et al., 2004).

Although immortality is a fundamental characteristic of most human cancers, the immortal phenotype alone is not sufficient to transform normal cells into their fully malignant counterparts (Morales, et al., 2003). As previously discussed, spontaneous transformation is a very rare occurrence as seen with hTERT immortalised fibroblasts that grew to over 300 population doublings and failed to transform (Morales, et al., 1999).

One of the anchorage independent colonies from P21d cell line was isolated and grown up and labelled P21d 0Gy (clone-a) as it was transformed but had not been irradiated. The transformed cells were most likely to be prostate epithelial cells and not another cell line infecting the culture as the cells grew in the presence of the two combined antibiotics, G418 and Puromycin. No other cell lines with this pattern of antibiotic resistance were being maintained in the lab at this time.

3.7.4 Plastic attached derived P21d clones

The cell line P21s never produced anchorage independent colonies whereas, the P21d cell line consistently produced increasing numbers of anchorage independent colonies after passage 9 with increasing passage number, Figure 3.1a. This tends to suggest that the P21d is a mixed culture of abnormal and normal cells and not exhibiting spontaneous transformation but the abnormal cells were proliferating faster than the normal cells and becoming the predominant part of the cell line (shown by increasing anchorage independent colony formation with increase in passage number). Therefore, it would have been anticipated that cloned cell lines from this early passage would have exhibited normal characteristics including no anchorage independent colony formation.

As seen in Figure 3.1a and b, P21d cell line produced anchorage independent colonies. This is a characteristic of potential transformed lines as anchorage independent growth is one of the most rigorous tests of cellular transformation in vitro and provides the best correlate to tumourigenic growth potential (Hamad, et al., 2002). The P21d cell line also exhibited abnormal morphology and a faster growth rate, Figure 3.2, 3.3. P21d cell line started showing anchorage independent growth at passage 9 and onwards, this suggests that the P21d cell line consisted of a mixture of normal and abnormal cells and that the abnormal cell population grew faster and took over the culture as the cell line was grown, Figure 3.1a. Therefore, if the early passage P21d cell line was plated on plastic dishes and the resulting colonies that grew up could be trypsined and grown up as new cloned cell lines. Assuming that P21d cell line is a mixed culture of normal and abnormal cells then some of the new cloned cell lines may be normal. This was done and seven cell lines were derived and propagated in culture. These cell lines were fully characterised for growth rate, screened for anchorage independent growth, c-Myc and cytokeratin expression, chromosome number, and percentage invasion across a Matrigel porous membrane, Table 3.2, Figure 3.4 and 3.5.

The P21d cloned lines from early passage P21d show abnormal characteristics such as anchorage independent growth, abnormal number of chromosomes per cell and invasion, Table 3.2, Figures 3.4 and 3.5. Therefore, they cannot be classed as normal cell lines derived from patient P21 prostate deep region but cloned tumour cell lines, Table 3.2. This suggests that all seven cell lines are abnormal (Hamad, et al., 2002; Ko, et al., 2003; Gu, et al., 2006). Invasion across an in vitro Matrigel porous membrane is a characteristic of cancer cell lines and has been used as a tool for predicating the ability of cancer cell lines to metastasise in in vivo conditions (Bello, et al., 1997). All of the cell lines showed invasion, Table 3.2. Metaphase spreads showed that the average copy number was more than diploid, Figure 3.4, Table 3.2. An abnormal large copy number of chromosomes is seen in prostate cancer cell lines and is associated with a neoplastic phenotype (Ko, et al., 2003; Gu, et al., 2004; Yasunaga, et al., 2001).

The cloned line g appeared to be the least abnormal of the cloned lines as it exhibited the least invasion, produced few anchorage independent colonies in agar and had the lowest abnormal number of chromosomes per cell (62 chromosomes), Table 3.2. However, it was not the slowest growing cell line, Figure 3.5.

All seven cloned cell lines exhibited abnormal characteristics including anchorage independent colony formation. Therefore, it can be concluded that the P21d cell line did not compose of a mixture of normal and abnormal cells but is an unstable population of abnormal cells. It thus might be possible that the patient P21 had prostatic intraepithelial neoplasia (PIN) in the deep region of the prostate and that a PIN region has been immortalised early on and propagated in culture, as PIN lesions in human prostates are very common in men with increasing age and thought to be the precursor of prostate cancer (Taichman, et al., 2007; Gelmenn, 2008; Stemmermann, et al., 1992).

Therefore, it could be concluded that the P21d cell line consists of a mixed population of cells from a PIN lesion that have been immortalised and as the cell line has grown up over time the more aggressive abnormal cells have dominated the culture shown by increasing anchorage independent colony formation. Many repeats all showed anchorage independent colony formation beginning at the same moment in culture, all at passage 9. This along with the seven cloned cell lines, which all showed abnormal characteristics and supports this hypothesis, shows that the anchorage independent colony formation is not a random event nor due to the immortalisation process, but a characteristic of the abnormal cells taken from patient P21.

This was an attempt to extract a normal cell line from P21d cell line. At early passage P21d cell line was thought to be a mixed culture of more normal than abnormal tumour cells. As the cells were plated at low doses the colonies seen were derived from a single cell and hence the

up to give cloned tumour lines, they can still be used as a novel tool to investigate prostate cancer.