Low-temperature plasma treatment induces DNA damage leading to necrotic cell death in primary prostate epithelial cells.

BACKGROUND: In recent years, the rapidly advancing field of low-temperature atmospheric pressure plasmas has shown considerable promise for future translational biomedical applications, including cancer therapy, through the generation of reactive oxygen and nitrogen species. METHOD: The cytopathic effect of low-temperature plasma was first verified in two commonly used prostate cell lines: BPH-1 and PC-3 cells. The study was then extended to analyse the effects in paired normal and tumour (Gleason grade 7) prostate epithelial cells cultured directly from patient tissue. Hydrogen peroxide (H2O2) and staurosporine were used as controls throughout. RESULTS: Low-temperature plasma (LTP) exposure resulted in high levels of DNA damage, a reduction in cell viability, and colony-forming ability. H2O2 formed in the culture medium was a likely facilitator of these effects. Necrosis and autophagy were recorded in primary cells, whereas cell lines exhibited apoptosis and necrosis. CONCLUSIONS: This study demonstrates that LTP treatment causes cytotoxic insult in primary prostate cells, leading to rapid necrotic cell death. It also highlights the need to study primary cultures in order to gain more realistic insight into patient response.

Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6.

BACKGROUND: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. METHODS: Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-(3)H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [(3)H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca(2+) levels. RESULTS: Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho). CONCLUSIONS: Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of PLD1 and increased turnover of EtnPGs. The phosphatidic acid formed will maintain a cancer phenotype through the regulation of mTOR. Ethanolamine released from cells may reduce Cho uptake, regulating the membrane PtdEtn:PtdCho ratio and influencing the action of PtdEtn-binding proteins such as RKIP and the anti-apoptotic hPEBP4. The work highlights a difference between LNCaP cells used as a model of androgen-dependent early stage PCa and androgen-independent PC3 cells used to model later refractory stage disease.

HDAC inhibitor confers radiosensitivity to prostate stem-like cells.

BACKGROUND: Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction. METHODS: Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2ß1integrin(hi)/CD133(+)), transit-amplifying (TA, α2ß1integrin(hi)/CD133(-)) and committed basal (CB, α2ß1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser. RESULTS: Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation. INTERPRETATION: Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

The immunosuppressive cytokine interleukin-4 increases the clonogenic potential of prostate stem-like cells by activation of STAT6 signalling.

Interleukin-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumour microenvironment of cancer patients, where concentrations correlate with the grade of malignancy. In prostate cancer, interleukin-4 has been associated with activation of the androgen receptor, increased proliferation and activation of survival pathways such as Akt and NF-κB. However, its role in therapy resistance has not yet been determined. Here we investigate the influence of interleukin-4 on primary epithelial cells from prostate cancer patients. Our data demonstrate an increase in the clonogenic potential of these cells when cultured in the presence of interleukin-4. In addition, a Phospho-Kinase Array revealed that in contrast to previously published work, signal transducer and activator of transcription6 (STAT6) is the only signalling molecule activated after interleukin-4 treatment. Using the STAT6-specific inhibitor AS1517499 we could confirm the role of STAT6 in increasing colony-forming frequency. However, clonogenic recovery assays revealed that interleukin-4 does not rescue the effects of either irradiation or docetaxel treatment. We therefore propose that although the interleukin-4/STAT6 axis does not appear to be involved in therapy resistance, it does play a crucial role in the colony-forming abilities of the basal cell population in prostate cancer. IL-4 may therefore contribute to disease relapse by providing a niche that is favourable for the clonogenic growth of prostate cancer stem cells.

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.

Expression of the N-myc oncogene in Wilms' tumour and related tissues.

Tissue samples and cell cultures from Wilms' tumour matched histologically normal kidney samples and EBV transformed B cells from the same patients, were analysed to detect changes in the structure and expression of the N-myc oncogene. The levels of expression of HLA class I and hypoxanthine guanine phosphoribosyl transferase were also measured in the various RNA preparations. Related tissue samples, from sources including congenital mesoblastic nephroma, paediatric neuroblastoma and a number of foetal tissues were also tested. Northern blot analysis indicated that the levels of N-myc were higher in Wilms' tumour tissues (with no parallel increase in gene copy number) compared to all other sources of material including foetal kidney. Particularly high levels of expression were observed in a number of the Wilms' tumours, several of which produced metastases. In situ hybridization, using [35S]-labelled RNA probes, confirmed that the high levels of N-myc RNA were present in the blastemal elements in the Wilms' tumour. All the tissue cultures, and tissue samples from other sources, except foetal brain and neuroblastoma, contained uniformly low levels of N-myc RNA.

Inactivation of the remaining allele of the WT1 gene in a Wilms' tumour from a WAGR patient.

A candidate gene (WT1) has recently been described for the 11p13 tumour-suppressor gene involved in the development of Wilms' tumour. This gene encodes a zinc finger protein which can bind to a specific DNA sequence. We have found a 226 base deletion in the mRNA from a unilateral Wilms' tumour, which would cause a frameshift that completely deletes the zinc finger domain. The tumour developed in a patient suffering from the WAGR syndrome, who had a constitutional 11p13 deletion, and so the 226 base deletion represents the inactivation of the remaining WT1 allele in the tumour. This provides further direct evidence that loss of function of WT1 is an essential step in the development of Wilms' tumour.

Alternative splicing of the human PTEN/MMAC1/TEP1 gene.

The human tumour suppressor gene PTEN/MMAC1/TEP1 encodes a lipid and protein phosphatase. Using RT-PCR, alternatively spliced forms of PTEN mRNA, encoding full-length PTEN and two forms of the protein truncated at the C-terminal end, were detected in normal human tissue. Cultured tumour and non-tumour cell lines show similar splicing patterns.

Detection of clonality in childhood B-lineage acute lymphoblastic leukaemia by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to study clonality in a group of children with B-lineage acute lymphoblastic leukaemia (ALL). Rearrangement of the immunoglobulin heavy chain gene (IgH) results in a hypervariable sequence known as the complementarity determining region III. This can be amplified by the PCR using one pair of consensus primers. The PCR product is highly clone-specific in both size and sequence. Successful amplification was achieved in 50 of 62 cases of B-lineage ALL studied (81%). Both DNA and RNA gave almost identical results. In contrast amplification was only achieved in 2 of 42 control cases (non-B-lineage leukaemias, normal and reactive marrows); these were both cases of T-ALL with IgH rearrangement on Southern blotting. The main advantages of this technique over Southern blot assessment of clonality are the short time to result and requirement for much less DNA allowing study of small samples eg cerebrospinal fluid and testicular biopsies. It is also generally more sensitive for the detection of a malignant clone in a polyclonal marrow cell population and forms the basis of techniques to study minimal residual disease (MRD).

miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of αv- and α6-Integrin Expression.

Altered microRNA (miRNA; miR) expression is associated with tumor formation and progression of various solid cancers. A major challenge in miRNA expression profiling of bulk tumors is represented by the heterogeneity of the subpopulations of cells that constitute the organ, as well as the tumor tissue. Here, we analyzed the expression of miRNAs in a subpopulation of epithelial stem/progenitor-like cells in human prostate cancer [prostate cancer stem cell (PCSC)] and compared their expression profile to more differentiated cancer cells. In both cell lines and clinical prostate cancer specimens, we identified that miR-25 expression in PCSCs was low/absent and steadily increased during their differentiation into cells with a luminal epithelial phenotype. Functional studies revealed that overexpression of miR-25 in prostate cancer cell lines and selected subpopulation of highly metastatic and tumorigenic cells (ALDH(high)) strongly affected the invasive cytoskeleton, causing reduced migration in vitro and metastasis via attenuation of extravasation in vivo. Here, we show, for the first time, that miR-25 can act as a tumor suppressor in highly metastatic PCSCs by direct functional interaction with the 3'-untranslated regions of proinvasive αv- and α6-integrins. Taken together, our observations suggest that miR-25 is a key regulator of invasiveness in human prostate cancer through its direct interactions with αv- and α6-integrin expression.

Preferential lectin binding to specific layers of rat oral epithelium and modification by enzyme pretreatment.

Three fluorescein-labeled lectins were shown to bind differentially to cell surfaces in different epithelial layers of rat oral mucosa regardless of the age or the site of origin of the tissue. Griffonia simplicifolia (GS-1-B4), specific for alpha-D-galactosyl end groups, labeled basal cells only; Ulex europeus (Ulex 1) specific for alpha-L-fucosyl groups labeled spinous cells; and Bandeiraea simplicifolia (BSII), specific for N-acetyl-D-glucosamine, labeled cornified cells. Pretreatment of sections with alpha-galactosidase completely abolished the staining of basal cells by GS-1-B4, but had no effect on the staining of spinous cells by Ulex 1. In contrast, alpha-fucosidase abolished the staining of spinous cells by Ulex 1 and caused staining of both basal and spinous cells by GS-1-B4. Neuraminidase and chondroitinase ABC produced results similar to one another, with staining of basal cells by GS-1-B4 and labeling of both basal and spinous cells with Ulex 1. beta-galactosidase, beta-glucuronidase, and testicular hyaluronidase did not affect the staining pattern of GS-1-B4 or Ulex 1, whereas chymotrypsin completely abolished any staining with either lectin. The results demonstrate a complex arrangement of cell surface carbohydrates in the epithelium of rat oral mucosa. The findings indicate a possible simplification in the spatial arrangements of cell surface carbohydrates during the differentiation of basal to spinous cells.

Molecular analysis of the interaction between HPV type 16 E6 and human E6-associated protein.

The complex formed between the human papillomavirus type 16 E6 protein and human E6-associated protein, which combine to ubiquitylate and degrade p53, has been studied by chemical crosslinking. Analysis of the interactions of proteins purified from Escherichia coli as well as proteins expressed in insect cells indicates that, while E6 has the capacity to form dimers, E6 and E6-associated protein interact as two monomers to form a heterologous dimer.

Targeting gene therapy for prostate cancer.

Gene therapy is rapidly emerging as a viable clinical strategy to treat prostate cancer. New developments, such as targeted expression of therapeutic genes, and viruses that are designed to selectively replicate in prostate cancer cells have led to vectors with improved safety, even in elderly male patients. This review describes the various different viral and non-viral strategies employed to date, with a summary of current clinical trials. The main focus of the review is a discussion of the need, and the potential methods that can be used for targeted expression of the therapeutic gene specifically to prostate tumours and metastases. This includes methods of abrogating vector transduction of non-specific tissues, enhancement of transduction into prostate tumour tissue, transcriptional control of the therapeutic gene and some examples of prostate cancer-specific therapeutic genes. We also consider the future of prostate cancer gene therapy and the factors that should be taken into account when designing clinical trials, in a field that is expected to impact on clinical management of a common tumour type.

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.

Androgen receptor localisation and turnover in human prostate epithelium treated with the antiandrogen, casodex.

In vitro models of normal and malignant human prostate are currently limited to a few well established cell lines that, with a single exception (LNCaP), fail to express the androgen receptor (AR) - a common characteristic of prostatic epithelium grown in culture. To investigate the molecular mechanism of action of the non-steroidal antiandrogen Casodex (bicalutamide) against wild-type AR, we have established a transient AR expression model in non-tumorigenic prostate cells of both epithelial and mesenchymal origin. In this model, both dihydrotestosterone and Casodex can effectively transport the AR protein into the nucleus of prostate cells. Whereas the natural ligand, dihydrotestosterone, stabilises the receptor, the AR is rapidly degraded at a nuclear location when the transfected cells are treated with Casodex. In contrast, whereas the mutant AR in the LNCaP line is also degraded on Casodex treatment over the same time period, its intracellular targeting is defective.

Targeting therapeutic gene expression to human prostate cancers.

Most current therapies against human carcinoma of the prostate are palliative rather than curative. Thus, there is a pressing need for new therapies directed against this common tumor, based on knowledge of the basic biology of the prostate, rather than by extrapolation of treatments from other tumor types. This review presents the various levels of prostate targeting which could be and have been applied, at the attachment, expression and therapeutic gene levels to eliminate prostate carcinoma cells in extraprostatic sites. To achieve optimal and safe targeting, a combination approach will be necessary, as there are few genes whose expression can provide absolute prostate specificity.

In vitro models to study cellular differentiation and function in human prostate cancers.

In Vitro Models to Study Cellular Differentiation and Function in Human Prostate Cancers. To augment the currently available models of human prostate cancer in vitro, we have established extended life-span epithelial cultures from biopsies of well-differentiated prostate cancers. The genetic identity of the target cells was assessed by allelotyping, using microsatellites located on chromosome 8p, and microdissection of tissues and primary cell cultures. Cells with an extended life span (PxE6) were derived by recombinant retrovirus infection to introduce the human papilloma virus E6 gene (epithelial cells). Immunophenotyping of the resultant cell strains confirmed retention of differentiated cell functions, and the genotype of the E6-expressing epithelial cells was stable, while SV40-immortalized cultures were more unstable, leading to tetraploidy. All PxE6 cells eventually senesced, but an immortalized epithelial culture, P4E6, was derived from one of the epithelial cultures. The properties of this cell line, which remains close to diploid, are similar to those of early prostate cancer cells, and it retains expression of many prostate-associated antigens, such as prostate-specific antigen (PSA).

Paternal origin of 11p15 duplications in the Beckwith-Wiedemann syndrome. A new case and review of the literature.

A boy suffering from the Beckwith-Wiedemann syndrome (BWS) was found to have partial trisomy of the short arm of chromosome 11 [46,XY,der(5)t(5;11)(p15.2;p14)]. Both his parents were phenotypically normal, but his father carried a balanced translocation between chromosomes 5 and 11 [46,XY,t(5;11)(p15.2;p14)]. DNA analysis of polymorphic markers on 11p15 confirmed the paternal origin of the duplicated material in the child. This case is the sixth report of paternal duplication of 11p15 in BWS. These results are discussed in relation to the possible role of genomic imprinting in BWS and in Wilms' tumor.

MS strain of type 2 herpes simplex virus produces necrotizing retinitis in mice.

Intracerebral inoculation of mice with the MS strain of type 2 herpes simplex virus (HSV-2) causes a brief encephalitis associated with multifocal central nervous system demyelination. Many of the mice develop unilateral or bilateral impairment of the pupillary light reflex. We have examined the development of ocular disease in inbred NIH mice inoculated intracerebrally with a low dose (10 pfu) of HSV-2 (MS). The resulting acute encephalitis was fatal in 30-50% of the mice. By 1 month after inoculation, the pupillary response to light was absent or impaired in approximately 80% of the surviving mice. Infectious virus could be isolated from the trigeminal ganglia and optic nerves from day 2 and from the eyes by day 4. Viral antigen was first immunohistochemically detectable in the optic nerves on day 5 and in the retinae on day 6. During the second week after inoculation up to half of the mice developed unilateral or bilateral necrotising retinitis associated with high titres of virus in the eyes and abundant viral antigen in the retinae. Electron microscopy confirmed the presence of viral particles in the retinae, in glia and degenerating neurons. No viral antigen was detected in the corneas and only rarely was antigen found in the ciliary body or iris. Infectious virus persisted longer in the eyes than in the trigeminal ganglia or optic nerves and could still be isolated from a few of the animals 2 weeks after inoculation. By 1 month the titres of virus within the eyes had fallen to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Latent infection with the MS strain of herpes simplex virus type 2 in the mouse following intracerebral inoculation.

Intracerebral inoculation of the MS strain of herpes simplex virus type 2 (HSV-2) into mice causes an acute encephalitis associated with multifocal demyelination and necrotizing retinitis. We have studied the distribution of latent virus in mice that had recovered from the acute encephalitis. Four weeks or longer after inoculation, HSV-2 could be recovered from the trigeminal ganglia of all mice examined by co-culture of explants in roller tubes. The virus could not be recovered from explants of retina or brain stem. HSV-2 latency associated transcript (LAT) was readily detected in the trigeminal ganglia by reverse transcriptase-PCR more than 4 months after inoculation. LAT was also demonstrated in the brain but this required nested PCR for consistent detection. Both LAT and ICP0 mRNA were detected in brain tissue during the acute encephalitis but, unlike LAT, ICP0 mRNA could not be amplified from the trigeminal ganglia or brain beyond 4 weeks after inoculation of the virus. In situ hybridisation with a double-stranded DNA probe to the ICP0/LAT overlap region of HSV-2 revealed signal in trigeminal ganglion neurons and occasional cells in the brain stem. These findings indicate that HSV-2 introduced by intracerebral inoculation becomes latent in the trigeminal ganglia and that transcription of LAT also persists within the brain.