DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

Retinoic acid represses invasion and stem cell phenotype by induction of the metastasis suppressors RARRES1 and LXN.

The mouse haematopoietic stem cell (SC) regulator Latexin (LXN) is the only known homologue of the retinoic acid receptor responder 1 (RARRES1) gene. Both genes lie adjacent on chromosome 3 and differ mostly by the presence of a transmembrane domain in RARRES1. Despite their homology, it is not known whether they possess similar regulatory mechanisms, cellular localization and function. Here, we identified RARRES1 and LXN as highly significantly downregulated genes in human prostate SCs, whose expression was induced by the pro-differentiation agent all-trans retinoic acid (atRA). AtRA induced expression in the most differentiated cells compared with the SC fraction, suggesting that this subpopulation was less responsive to atRA. Small interfering RNA suppression of RARRES1 and LXN enhanced the SC properties of primary prostate cultures, as shown by a significant increase in their colony-forming ability. Expression of both RARRES1 and LXN was co-ordinately repressed by DNA methylation in prostate cancer cell lines and inhibition of RARRES1 and LXN increased the invasive capacity of primary prostate cultures, which also fully rescued an inhibitory effect induced by atRA. Moreover, we showed that RARRES1 and LXN reside within different sub-cellular compartments, providing evidence that RARRES1 is not a plasma membrane protein as previously supposed but is located primarily in the endoplasmic reticulum; whereas LXN was detected in the nucleus of prostate epithelial cells. Thus, LXN and RARRES1 are potential tumour suppressor genes, which are co-ordinately regulated, SC-silenced genes functioning to suppress invasion and colony-forming ability of prostate cancer cells; yet the proteins reside within different sub-cellular compartments.

A rare cause of scrotal swelling: transitional cell carcinoma of the bladder presenting as a testicular metastasis.

A 72-year-old Caucasian male who presented with haematuria in July of 2000 was found to have a large left-sided bladder tumour. He underwent a transurethral resection of the tumour and surveillance program. In October 2008 he underwent a transurethral resection of the prostate (TURP). Histology of the prostatic chippings showed poorly differentiated TCC with prostatic invasion. A CT of his chest abdomen and pelvis revealed no lymph node involvement or metastatic spread. He therefore underwent a cystoprostato-urethrectomy with ileal conduit formation, in December 2008. In May 2010 the decision was made to perform a left inguinal orchidectomy as he presented with a craggy mass of his left testis, and there were clinical concerns that this was a tumour. Histology revealed that the left testis had been wholly replaced by a tumour. Taking into account his previous urological history, the features of this tumour are consistent with metastatic TCC, which is very rare.

Human prostate cell lines from normal and tumourigenic epithelia differ in the pattern and control of choline lipid headgroups released into the medium on stimulation of protein kinase C.

BACKGROUND: Expression of protein kinase C alpha (PKCα) is elevated in prostate cancer (PCa); thus, we have studied whether the development of tumourigenesis in prostate epithelial cell lines modifies the normal pattern of choline (Cho) metabolite release on PKC activation. METHODS: Normal and tumourigenic human prostate epithelial cell lines were incubated with [(3)H]-Cho to label choline phospholipids. Protein kinase C was activated with phorbol ester and blocked with inhibitors. Choline metabolites were resolved by ion-exchange chromatography. Phospholipase D (PLD) activity was measured by transphosphatidylation. Protein expression was detected by western blotting and/or RT-PCR. Choline uptake was measured on cells in monolayers over 60 min. RESULTS: Normal prostate epithelial cell lines principally released phosphocholine (PCho) in contrast to tumourigenic lines, which released Cho. In addition, only with normal cell lines did PKC activation stimulate Cho metabolite release. Protein kinase C alpha expression varied between normal and tumourigenic cell lines but all showed a PKCα link to myristoylated alanine-rich C kinase substrate (MARCKS) protein. The five cell lines differed in Cho uptake levels, with normal PNT2C2 line cells showing highest uptake over 60 min incubation. Normal and tumourigenic cell lines expressed mRNA for PLD1 and PLD2, and showed similar levels of basal and PKC-activated PLD activity. CONCLUSIONS: The transition to tumourigenesis in prostate epithelial cell lines results in major changes to Cho metabolite release into the medium and PKC signalling to phosphatidylcholine turnover. The changes, which reflect the metabolic and proliferative needs of tumourigenic cells compared with untransformed cells, could be significant for both diagnosis and treatment.

Prostate cancer stem cells: a target for new therapies.

Prostate cancer is now a common disease in men over 50 years of age. Medical therapies for prostate cancer are based on discoveries from the mid-twentieth century, and in the long term are rarely curative. Most treatments are directed towards an androgen receptor-expressing, highly proliferative target cell, which does indeed form the vast majority of cells in a prostate tumour. However, by invoking the existence of a cancer stem cell which, like normal epithelial stem cells in the prostate, does not express androgen receptor and is relatively quiescent, the observed resistance to most medical therapies can be explained. The phenotype of the prostate cancer stem cells is that of a basal cell and cultures derived from cancers, but not benign tissues, express a range of prostate cancer-associated RNAs. Furthermore, stem cells purified on the basis of alpha2beta1 high integrin and CD133 cell surface antigen expression, from an established culture of Gleason 4 (2+2) prostate cancer (P4E6), were able to form multiple intraprostatic tumours in nude mice when grafted orthotopically in a matrigel plug containing human prostatic stroma. The final tumours reexpressed androgen receptor and displayed a histology similar to that of a Gleason 4 cancer.

The role of percutaneous nephrostomy in malignant ureteric obstruction.

OBJECTIVES: Uraemia as a result of malignant ureteric obstruction is a recognised event in those with advanced malignancy, usually of pelvic origin, which, if left untreated, is quickly a terminal event. Palliative decompression of the obstructed urinary system, either by percutaneous nephrostomy (PCN), ureteric stent or a combination of both is a recognised method of improving renal function, with presumed low morbidity. The aims of the study were to assess whether PCN placement in malignant ureteric obstruction provided any additional survival benefit or patient morbidity. PATIENTS AND METHODS: The case notes of 32 patients with a mean age of 68.1 years (16 male, 16 female) who underwent PCN drainage for malignant ureteric obstruction were retrospectively analysed. Data on the site of primary malignancy, mode of presentation, improvement in renal function, median survival, conversion to internal ureteric stents and intervention-related complications were collected for analysis. RESULTS: The median survival following PCN insertion was 87 days and was unrelated to the patient's age and renal function. Those patients with primary underlying gynaecological malignancies appeared to survive almost 4 times as long as those with underlying primary bladder cancer. Renal function took a mean of 16.8 days to reach a nadir. Almost 79% of patients were able to be discharged from hospital--each patient, however, being re-admitted back to hospital on average 1.6 times prior to their death through PCN or internal ureteric stent related events. Retrospective "useful quality of life" was seen in less than half of the patient cohort. CONCLUSIONS: In the presence of malignant ureteric obstruction, palliative percutaneous urinary diversion may be performed and is effective in improving renal function. However, long-term survival is limited and should, therefore, be performed only when the views and wishes of the patient and carers are taken into account and if there is a definitive treatment plan available for the patient as quality of life can be suboptimal.

The changing practice of transurethral prostatectomy: a comparison of cases performed in 1990 and 2000.

OBJECTIVES: Transurethral resection of the prostate (TURP) is considered by many to be the 'gold standard' treatment for benign prostatic enlargement. However, with the relatively recent introduction of pharmacological and other surgical treatment modalities, the performance of TURP appears to be in decline. METHODS: A retrospective casenote analysis of 200 patients who underwent TURP in 1990 and the year 2000 with the aim of identifying changes in the incidence and practice of TURP. RESULTS: There was a decline in the number of TURPs performed of 31.6% over the 10-year period, with more being carried out because of urinary retention. In 2000, the patient was older and the operative procedure took statistically longer than 10-years earlier, but the weight of prostate tissue resected, patient satisfaction and complication rates were similar. CONCLUSIONS: At present, TURP is in decline, with urinary retention being the commonest indication. The population at present is older but this does not carry additional co-morbidity. The weight of resection has not altered, although surgery currently takes longer to perform.

Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture.

To reproduce the structural and functional differentiation of human prostatic acini in vivo, prostatic epithelial and stromal cells derived from human primary cultures were cocultured in Matrigel. In the absence of stroma and serum, epithelial spheroids composed of solid masses of stratified and cuboidal cells formed. Outer cells of the spheroid expressed cytokeratins 1, 5, 10, and 14, whereas the inner cells expressed cytokeratin 18. The addition of 2% serum induced formation of a lumen surrounded by a layer of one or two cuboidal and columnar epithelial cells. The further addition of stromal cultures, dihydrotestosterone, and estrogen induced polarization of the epithelium and increased spheroid-forming efficiency. Epithelium expressed either cytokeratin 18 alone or additionally cytokeratins 1, 5, 14, and 10. All spheroid epithelium expressed prostate-specific antigen and prostate-specific membrane antigen. Androgen receptor was only detected in the presence of stroma, serum, and hormones. Thus, development of a functional and morphologically correct prostate gland in vitro is dependent on extracellular matrix, steroid hormones, and factors from stromal cells and serum.

In vitro modelling of epithelial and stromal interactions in non-malignant and malignant prostates.

To study the effects of stromal epithelial cell interactions on prostate cancer metastasis, we have used primary human prostatic stromal cells derived from malignant and non-malignant tissues and established epithelial cell lines from normal (PNT1a and PNT2-C2) and tumour (PC-3, DU145 and LNCaP) origins. The effects of stromal cells on epithelial cell growth were studied in direct and indirect (using culture inserts) co-culture and by exposure to stromal cell-conditioned medium (assessed by MTT assay). The influence of stromal cells on epithelial cell invasion was measured using matrigel invasion chambers and on epithelial cell motility using time lapse microscopy. Results indicated that epithelial cell line growth was similarly unaffected or inhibited by stromal cells derived from malignant (n = 8) or non-malignant tissue (n = 8). In contrast, PNT2-C2 and PC-3 cells were found to be the least and the most invasive and motile epithelia respectively. Stromal cultures enhanced the invasion of both epithelial cells, but no differences were observed between the use of malignant and non-malignant tissues. All stromal cultures modestly stimulated PNT2-C2 motility but displayed a greater stimulation of PC-3 cell motility, while stromal cells derived from malignant tissue stimulated PNT2-C2 and PC-3 cell motility more than stromal cultures from non-malignant tissues.

Allelic imbalance within the E-cadherin gene is an infrequent event in prostate carcinogenesis.

By exploiting two single nucleotide polymorphisms (SNPs) located within the E-cadherin gene, at 16q22, we have determined the frequency of allelic imbalance at this proposed tumor suppressor locus in a series of human prostatic carcinoma DNA samples. Whereas results with seven highly polymorphic microsatellite markers flanking the E-cadherin locus confirmed the existence of three separate loci on chromosome 16, at which allelic imbalance increased with increasing loss of tumor cell differentiation, no allelic imbalance within the E-cadherin gene was detected either by single-strand conformational polymorphism analysis or by direct sequencing. We conclude that the loss of E-cadherin function observed in prostate cancer is not a result of allelic deletion. Genes Chromosomes Cancer 27:104-109, 2000.

Precise microdissection of human prostate cancers reveals genotypic heterogeneity.

To determine the incidence of genetic heterogeneity in primary prostate cancer, we have microdissected 125 tumor and mesenchymal foci from 18 patient biopsies and analyzed the DNA for loss of heterozygosity using PCR microsatellite markers. In 100% of patients with genetic lesions on chromosome 8p, there was evidence for intratumoral genetic heterogeneity. There was also a low but significant incidence of loss of heterozygosity in mesenchymal tissue. Our results show that phenotypically similar tumor foci can have different genotypes and provide evidence for the multifocality of tumor development in the prostate.

A hot spot for p53 mutation in transitional cell carcinoma of the bladder: clues to the etiology of bladder cancer.

Twenty-eight transitional cell carcinomas of the bladder, grade 2 or 3, were analyzed for the presence of p53 mutations. Thirteen tumors were found to contain 14 mutations. These were all base substitution mutations, of which nine were GC-->AT transitions (three at CpG sites). The remaining five mutations were transversions (three GC-->CG, one GC-->TA, and one AT-->TA). Four of the mutations were found at codon 280. A comparison with other studies of bladder tumors reveals that a region encompassing codons 280 and 285 represents a hot spot for p53 mutation in bladder cancer. The 280/285 hot spot lies within two purine-rich sequences that may provide some clues to the identity of potential bladder carcinogens. A comparison of mutations from bladder tumors of smokers and nonsmokers reveals no significant differences.

Analysis of prostate tissue DNA for the presence of human papillomavirus by polymerase chain reaction, cloning, and automated sequencing.

We have analysed the DNA from 24 prostate tissue biopsies, spanning a range of Gleason grading from benign to grade 5 and mixed randomly with cervical cancer samples of known human papillomavirus (HPV) status, for the prevalence of HPV DNA, in a double-blind study to ensure complete objectivity. Polymerase chain reactions (PCR) were performed using general E1 open reading frame primers for HPV under low stringency conditions, in addition to reactions containing primers specific for HPV16, E2, and E6 open reading frames under higher, more stringent PCR conditions. The presence of cellular DNA was verified by the use of primers for hypoxanthine guanine phosphoribosyl transferase. DNA bands were not detected in the prostate biopsies using the HPV16-specific primers under high-stringency PCR conditions, however a predominant band in the 400 bp region was observed in 15 of the prostate biopsies using the general primers and the low annealing temperature of 40 degrees C. This fragment was excised and cloned into the pT7 blue vector and the sequence of the insert determined. Although the cloned sequences initiated and terminated with the two authentic PCR primers, they did not contain a significant HPV-related open reading frame. Our results indicate that HPV type 16 and closely related types, as detected by the general primer pair, are unlikely initiators of prostate carcinogenesis within our population.

Co-ordinated changes in expression of cell adhesion molecules in prostate cancer.

The expressions of E-cadherin, the integrin subunits beta 1, beta 2, beta 3, CD44 and alpha-catenin were studied in parallel by immunohistochemistry in a series of 40 prostate biopsies comprising one normal, 11 benign prostatic hyperplasia (BPH), and 28 prostatic adenocarcinomas. As reported by others, there was a consistent loss of E-cadherin expression with increasing tumour grade and de-differentiation. However, a significant proportion of losses occurred at earlier grades than previously reported. The parallel nature of this study showed, for the first time in human prostate carcinoma, a reciprocal expression pattern of E-cadherin and beta 1 integrin in the higher grades of prostate cancer. A reciprocal expression pattern was also found for E-cadherin and CD44 between moderately and poorly differentiated tumours. alpha-Catenin expression was downregulated only in those cells which had previously lost E-cadherin expression, and beta 2 and beta 3 integrin were rarely expressed in prostate tumours. A loss of expression of the luminal epithelial specific keratins CK8 and CK18 was also observed in advanced stage, poorly differentiated carcinomas.

Molecular screening of multifocal transitional cell carcinoma of the bladder using p53 mutations as biomarkers.

Thirteen of 28 patients (46%) with grade 2-3 multifocal transitional cell carcinoma (TCC) of the bladder were found to have p53 mutations using DNA sequence analysis. These were subsequently utilized as tumor-specific biomarkers. Analysis of 17 episodes of recurrence from five of the patients revealed that all but one carried the identical mutation to the primary tumor. Thirty urine samples were collected, at initial diagnosis and during follow-up screening, from eight patients with mutations over a period of 24 months. Sequence analysis of PCR products generated from DNA extracted from the urine sediments was carried out. The p53 mutation seen in the primary tumors was detectable in 24 of 30 urine samples. The remaining six cases coincided with a negative cystoscopic examination. Interestingly, 6 of the 24 urine samples in which mutations were detectable also coincided with negative cystoscopy. The results are consistent with: (a) monoclonality of multifocal TCC; (b) the spread of TCC through a seeding mechanism; and (c) the long-term persistence of tumor cell clones (up to 97 months) within the bladder, even in the absence of obvious tumor growth.

A microdissection approach to detect molecular markers during progression of prostate cancer.

To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate.

Quantification of prostatic cancer metastatic disease using prostate-specific antigen.

The serum prostate-specific antigen (PSA) of 58 men with benign prostatic hypertrophy (BPH) and 17 men with carcinoma of the prostate (CaP) was correlated with the weight of prostatic tissue resected at transurethral prostatectomy (TURP). A significant correlation was identified between the weight of resected BPH tissue and the serum PSA (p less than or equal to 0.001; r = 0.54). No such correlation was seen in the CaP patients. By arbitrarily dividing the serum PSA by the prostate weight, it was possible to devise an index. This index corrected PSA in relation to prostatic size and unlike PSA in isolation did not differ significantly between normal controls and those with BPH. The index in CaP was significantly greater than that of either controls or BPH (p less than or equal to 0.001). Furthermore the index of metastatic CaP (M1) was significantly higher than that of nonmetastatic disease (MO) (p = 0.05). The higher index found in CaP would seem to be related to the bulk metastatic tumor, either manifest or occult. Comparing the index of CaPs to that found in normal and benign disease (a constant) offers a possible means of estimating the extent of local and metastatic tumor mass.

Life with urostomy.

The quality of life of urostomy patients was assessed by direct interview. Specific problems, such as urine leakage, were documented and the size, site and quality of the stoma were assessed. The study included 41 patients; 83% noted an improvement in the quality of life since their urinary diversion and 90% were still able to work or continue to perform household duties. Urine leakage was the commonest problem and this was unrelated to the length of the stoma or the build of the patient. More important factors were the correct siting of the stoma and the avoidance of parastomal herniation. All patients emphasised the importance of the stoma therapist.

Inverted papilloma of the ureter with malignant change.

Three patients with inverted papilloma of the ureter are described. A range of histological features was seen, including one showing malignant change. This condition, which is probably more common than previously thought, can be successfully treated by conservative surgery and close follow-up.