P53 expression is associated with malignant potential in xenograft tissues of a fibrosarcoma mouse model.

BACKGROUND: The expression of wild-type and mutant p53 was studied in two fibrosarcoma cell lines in a mouse xenograft model. MATERIALS AND METHODS: Human cell lines HT1080 and Hs913(D)T were implanted in athymic mice via intramuscular (i.m.) or subcutaneous (s.c.) routes. After eight weeks, liver, lung and primary inoculation sites were harvested. Sections were stained using two methods: a) haematoxylin and eosin to detect tumour at implantation site, liver and lung; b) immunohistochemistry using monoclonal antibodies to detect expression of wild-type (wt) and mutant p53. RESULTS: Both cell lines had similar implantation rates via either route but Hs913(D)T had a higher metastatic rate than HT1080. The Hs913(D)T cells exhibited greater expression of mutant and wild-type p53 than the HT1080 cells. CONCLUSION: The expression of wild-type and mutant p53 is associated with a cell line of greater malignant potential. The inoculation route does not affect primary tumour uptake or metastatic rate.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo.

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer.

BACKGROUND: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. METHODS: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. RESULTS: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. CONCLUSIONS: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.

Downregulation of KAI1 mRNA in localised prostate cancer and its bony metastases does not correlate with p53 overexpression.

Recent data have proposed that transcription of the KAI1 metastasis suppressor gene is directly mediated by p53 and that loss of KAI1 expression in advanced prostate cancer is simply due to loss of p53 function after mutation. To investigate this possibility, we have examined KAI1 mRNA (by in situ hybridisation) and p53 protein expression (by immunohistochemistry) as an indicator of wildtype or mutant p53, in a series of 77 paraffin-embedded prostate tissue samples, including post-mortem normal prostates (2), benign prostatic hyperplasia (10), localised cancer (grades 4-6, 25; grades 7-9, 21) and prostate-derived bony metastases (19). Overall, we confirmed that expression of KAI1 mRNA decreased from normal tissue, through localised cancer to bony metastases (P=0.055, tending to significance), while levels of p53 staining significantly increased with cancer progression (P=0.046). These were consistent with the possibility that loss of p53 function might be responsible for loss of KAI1 mRNA. However, by close examination of KAI1 and p53 in adjacent tissue sections, we found no correlation between decreased levels of KAI1 mRNA and overexpression of p53 protein (P=0.497). In addition, high levels of KAI1 mRNA could be identified in samples irrespective of p53 staining. Our data suggest that mutation of p53 is independent of the loss of KAI1 mRNA, and do not support a role for p53 in regulating the expression of KAI1.

Premature discharge in a community hospital.

AIM: To determine the size of the problem of premature discharge in a community hospital (CH) and to ascertain the reasons for it. METHOD: A retrospective review of all admissions in year 2000 which resulted in premature discharge i.e. discharge within one week of admission, was conducted in a community hospital, St Luke's Hospital for the Elderly. Information collected on the selected cases included biodata, reason for CH stay, admitting diagnosis, source of admission, duration of stay and reason for terminating stay prematurely. For cases which required acute hospital transfer or ended in death in the CH, the type and day of onset of the respective medical problems were documented. RESULTS: Out of 924 admissions in year 2000, 12% resulted in premature discharge. Within this category of patients, 54% were discharged within the first three days and median duration of stay was three days. Majority of the admissions were for rehabilitation (83%) and respite care (15%). Neurological (60%) and orthopaedic (18%) problems constituted the bulk of the admitting diagnoses. The main reason for premature discharge was acute hospital transfer (90%) for medically unstable patients and those with unresolved medical problems. CONCLUSION: Premature discharge in the CH is an important issue and the greater cause lies in the need to transfer medically unstable patients or patients with unresolved medical problems back to the acute hospital. Stricter enforcement of admission criteria into CHs, increased vigilance on the part of acute hospitals and implementation of subacute care in CH can be solutions to the problem.

Methylation of a CpG island within the promoter region of the KAI1 metastasis suppressor gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines.

The molecular basis for downregulation of the KAI1 metastasis suppressor gene in invasive and metastatic human cancers is unknown. We have used bisulphite methylation analysis of DNA from paraffin-embedded invasive bladder tumour samples and from bladder cancer cell lines to determine if hypermethylation of a CpG island within the KAI1 promoter is responsible for this effect. Representative invasive tumour cell lines were also exposed to 5-aza-2-deoxycytidine. We found no evidence for hypermethylation of the CpG island and suggest that mechanisms other than promoter hypermethylation are responsible for reduced KAI1 expression in invasive bladder tumours and tumour cell lines.

Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen.

It has been observed that immunoreactivity in paraffin sections decreased during storage. In this study, stored paraffin sections from both biopsy material and cultured cells were assessed for changes in immunoreactivity, using color-based image analysis to quantitate extent and intensity of the stainings. For seven of the 11 antibodies studied, storage at 20 degrees C for 16 weeks reduced the extent of immunostaining compared with that of freshly cut sections. Furthermore, increased storage temperatures resulted in a progressive loss of immunoreactivity. After 2 weeks of storage, at both 4 degrees C and 20 degrees C, p53 protein- and MIB1-antigen expression was significantly reduced regarding extent and intensity. The extent of the immunoreactivity reduced more for p53 protein than for MIB1 antigen, but the intensity did not. Boric acid was used for antigen retrieval on sections stored for 12 weeks at 20 degrees C. For both p53 protein and MIB1 antigen, this resulted in an extent and intensity of immunostaining equal to or higher than (MIB1) that obtained in freshly cut sections, using citrate buffer. Staining of cultured cells confirmed the results from biopsy material on the influence of storage temperature. Fixation time only marginally influenced the storage-related decrease in immunoreactivity. In conclusion, storage of paraffin sections leads to a varying degree of decreased immunoreactivity for several antibodies. The degree is at least partly dependent on storage time and temperature but not fixation time. However, this may be compensated for by optimizing the antigen retrieval protocol.

Relationship between expression of the KAI1 metastasis suppressor and other markers of advanced bladder cancer.

Expression of a newly described inhibitor of tumour metastasis, KAI1, was examined in bladder cancer progression and compared with the expression of p53 and pRb, which are markers of advanced disease. KAI1 mRNA (by in situ hybridization) and protein levels (by immunohistochemistry) were examined in 135 paraffin-embedded bladder tissue sections. Significant decreases in KAI1 mRNA and protein levels were detected between normal and tumour tissue (p<0.001 and p=0.026, respectively), and between non-invasive and invasive tumours (p=0.046 and p<0.001, respectively). Loss of KAI1 protein expression was accompanied by a shift in staining pattern from a uniform distribution to a weaker, membranous or heterogeneous pattern. Normal tissue and low-grade tumours showed little p53 protein staining. High level staining (indicative of mutant p53) was associated with increased grade in non-invasive tumours (p=0.031) but was not significantly higher in invasive tumours. Whilst p53 protein staining increased with malignant progression and KAI1 mRNA expression decreased, there was no significant correlation between the two patterns (p=0.33, adjusted for group, p=0.18) or when only cancer samples were analysed (p=0.065, adjusted for group, p=0.26), even when taking into account overexpression of MDM-2 protein as a pathway for inactivation of p53. There was no correlation between loss of KAI1 mRNA expression and gain of abnormal pRb staining (p=0. 30, or adjusted for tumour samples only, p=0.59). These results suggest that loss of KAI1 expression is associated with invasive bladder cancer, but is not related to mutation of p53 or to loss of normal pRb expression.

Beta-human chorionic gonadotropin in semen: a marker for early detection of prostate cancer?

BACKGROUND AND PURPOSE: The beta subunit of human chorionic gonadotropin (beta-hCG) has been detected in prostate cancer by immunologic and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Recently, prostate cells have been detected in human ejaculate. This study aimed to determine if beta-hCG could be detected by RT-PCR from prostatic mRNA isolated from semen, thus providing a noninvasive procedure for prostate cancer detection. RESULTS: Expression of beta-hCG in prostate cancer was confirmed by immunohistochemistry methods. The protein was associated with low-grade disease: Gleason Score 4 through 7 (N = 26; 69%) but not high-grade (Gleason 8 or 9) or metastatic (lymph node) disease (N = 12; 8%). Normal prostate tissue was negative for beta-hCG (N = 14). The beta-hCG RT-PCR was performed on RNA extracted from seven human prostate cancer cell lines, which showed variable expression of beta-hCG mRNA. Semen was collected from patients suspected of having carcinoma of the prostate (N = 94) and from volunteers who were under the age of 30 years and had no family history of prostate cancer (N = 9). mRNA for beta-hCG was detected in the ejaculates of 12% of the patients with confirmed prostate cancer (N = 42) but not in any patients found to be negative for cancer (N = 52). Expression of beta-hCG mRNA was found in 22% of the control samples. CONCLUSIONS: The beta-hCG protein is expressed in low-grade prostate cancer and can be detected by RT-PCR in both prostate cancer cell lines and human ejaculate. However, the low percentage of detection in ejaculate suggests that beta-hCG in semen does not provide a useful marker for early prostate cancer detection.

Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

Caffeine-increased radiosensitivity is not dependent on a loss of G2/M arrest or apoptosis in bladder cancer cell lines.

PURPOSE: Bladder cancer cell lines UCRU-BL-13, UCRU-BL-17/2 and UCRU-BL-28, with differing p53 status and molecular responses to irradiation, were used to investigate possible mechanisms for caffeine-induced radiosensitization. MATERIALS AND METHODS: After treatment with caffeine and exposure to X-radiation, radiosensitivity was determined by clonogenic assay. Cell-cycle arrest and apoptosis were measured by flow cytometry. RESULTS: Both BL-13 and BL-28 cells (each expressing p53 with a wild-type sequence) fail to arrest at the G2 checkpoint after radiation, but nevertheless caffeine did induce radiosensitization. In contrast, in BL-17/2 cells (expressing p53 with a point mutation in codon 280), caffeine treatment abrogated the radiation-induced G2 arrest but was not accompanied by radiosensitization. No effects on radiosensitivity were seen in RT112 cells (expressing a functionally defective p53) at low caffeine doses (2 mM), but at higher doses (4 mM and 10 mM) caffeine caused both abrogation of radiation-induced G2 arrest and radiosensitization. In none of the cell lines examined did caffeine treatment and/or irradiation result in apoptosis. CONCLUSIONS: In contrast with previous studies, the data suggest that radiosensitization induced by caffeine is not dependent on abrogation of G2 arrest or the induction of apoptosis, and is not selective for cells expressing p53 proteins with mutations.

DNA-Flow cytometric analysis of bladder TCC using paraffin-embedded tissues.

A retrospective study of DNA flow cytometry (FCM) in paraffin-embedded tissues of urinary bladder transitional cell carcinoma (TCC) was performed on 239 biopsy samples taken from 81 patients in the period from 1984 to 1994. 210 (87%) were analysable. Of these samples 21 patients had multiple biopsies taken from large tumours and/or bladder mucosa showing an endoscopically normal appearance. DNA-FCM results have been evaluated comparing ploidy and histopathological grade, clinical stage and different clinical status, i.e., first diagnosis, recurrence and patients who died from bladder cancer. Our results indicate that 'diploid' FCM correlated with a better prognosis, whilst DNA aneuploid correlated with malignancy and a poorer prognosis. There was a trend to an increasing incidence of DNA aneuploidy as the grade of the tumour rose and the proportion of biopsies with aneuploidy was significantly higher in malignant tissue samples, recurrences and in biopsies from patients who died from TCC than in other groups. In 12 patients from whom several biopsies were obtained, samples from recurrences had significantly higher DNA aneuploidy than those from the first diagnosis.

Sources of variability in the determination of micronuclei in irradiated peripheral blood lymphocytes.

Following a dose of 4 Gy 250 kVp X-rays to the blood lymphocytes from three healthy donors the frequency of micronuclei was enumerated by three scorers. Eight slides were prepared from each blood sample with 1000 cytokinesis-blocked binucleate cells being scored for micronuclei and a recount undertaken 4 weeks later. Seven possible sources of variation were investigated: differences between donors, scorers, the four stages of sample preparation and time. A complete factorial analysis indicated that the major sources of variation were differences between donors and differences between scorers. One scorer identified fewer micronuclei than did the other two scorers in all slides of the irradiated blood samples and gave wide and inconsistent results for baseline counts. These results suggest that the performance of scorers should be screened before undertaking analysis of micronuclei. The most striking result, however, was the consistent performance of the scorers on the repeat counts compared with the very large differences between the scorers.

Higher expression of oncoproteins c-myc, c-erb B-2/neu, PCNA, and p53 in metastasizing colorectal cancer than in nonmetastasizing tumors.

BACKGROUND: Expression of individual oncogenes may predict outcome in patients with metastatic colorectal cancer (CRC). We studied the oncogene profile in the tumors of patients with CRC and assessed their value as predictors of liver metastases. METHODS: The oncoproteins c-myc, c-erbB-2/neu (c-neu), PCNA and p53, were measured by immunohistochemistry in sections of metastasizing human CRC (n = 34) and their liver secondaries as well as in sections of nonmetastasizing human CRC (n = 25). RESULTS: The metastasizing primary CRC expressed proliferating-cell nuclear antigen (PCNA), c-neu, and c-myc at significantly higher levels than the nonmetastasizing primary cancer, p53 was also overexpressed in the metastatic group compared with the nonmetastasizing CRC, but this difference was not significant. The frequency of expression of all these markers was similar in the metastasizing primary CRC and the liver secondaries from the same patients. There was no correlation between the expression of the individual markers and histological grade, DNA ploidy, and subsequent local recurrence and lung metastasis and survival. However, when both groups were assessed together, positive expression of c-myc was more likely to occur in poorly differentiated tumors, whereas PCNA expression increased with more advanced Dukes stages. CONCLUSION: These results suggest that the overexpression of c-myc, c-neu, PCNA, and p53 may occur in CRC that are likely to metastasis to the liver.

DNA flow-cytometric analysis in colorectal cancer: a comparison of metastasizing and non-metastasizing tumours.

The most common cause of death in patients with colorectal cancer is metastatic liver disease. In order to identify patients at a high risk of developing hepatic secondaries from colorectal cancers, DNA content was measured in metastasizing colorectal primaries (Group I, n = 32) as well as in their subsequently resected liver secondaries and in sections of non-metastasizing colorectal cancers (Group II, n = 25). A modified interpretation system involving both a DNA index and percentage of cycling cells (those in S and G2 + M phases) was developed. DNA content was measured in paraffin-embedded sections by flow cytometry using internal controls (human peripheral blood mononuclear cells) and non-malignant tissue controls (19 patients with diverticular disease). In Group I there were significantly more tumours with both abnormal ploidy (aneuploid or abnormal tetraploid peak) and > 15% cycling cells compared with Group II (Chi-squared; P = 0.034). The combination of abnormal ploidy and > 15% cycling cells was superior to Dukes' classification for identifying metastasizing tumours (Logistic Regression; P = 0.047). However, it was not possible to discriminate between the two groups using either DNA ploidy or the percentage of cycling cells alone. The metastasizing colorectal cancers exhibited similar DNA ploidy characteristics and had a similar percentage of cycling cells compared with their liver metastases. These results suggest that tumour DNA ploidy plus the percentage of cycling cells may predict the development of liver metastases and thus survival in patients with colorectal cancer.

The prognostic significance of tumor-associated markers p53, HER-2/neu, c-myc, v-H-ras, PCNA and EGFr of local and distant recurrence in localized human prostatic adenocarcinoma.

The intracellular expression of the gene products of tumor-associated markers p53, proliferative cell nuclear antigen (PCNA), HER-2/neu, c-myc, H-ras, and epidermal growth factor receptor (EGFr) in 86 cases of localized prostatic adenocarcinoma was investigated immunohistochemically in formalin-fixed paraffin-embedded tissue sections after pretreatment with a novel antigen retrieval buffer. A scoring system was devised to assess strength, pattern, and combined strength/pattern of immunostainings in the nucleus and cytoplasm for each immunomarker. The results were evaluated to determine whether overexpression of the gene products in the nucleus and cytoplasm was predictive of local and/or distant tumor recurrence and whether their expression was associated with known clinical prognostic factors. There was no significant relation between p53, PCNA, HER-2/neu, c-myc, and H-ras protein expression with risk of recurrence. EGFr expression showed a trend of increasing risk of tumor recurrence with higher composite score. Analysis of the association with other known prognostic factors in prostatic adenocarcinoma showed that PCNA was significantly correlated with tumor stage while H-ras and HER-2/neu were marginally correlated with prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) pretreatment serum levels, respectively. Together our findings suggest that overexpression of these intracellular oncoproteins in the tumor cells may not play an important role in determining whether prostatic tumors are likely to recur in localized prostatic adenocarcinoma.

Cloning of estrogen-responsive messenger RNAs in the T-47D human breast cancer cell line.

A complementary DNA library has been constructed from the polyadenylated mRNA of steroid-deprived T-47D cells which had been restimulated with estrogen for 24 h. Screening of 15,000 recombinants by sequential rounds of colony, Southern and Northern blot differential hybridization has identified eight different clones which vary in abundance and are stimulated between 2.5- and 8-fold by estrogen. Whereas five recombinants hybridize to single mRNA sequences, two clones, pSyd 2 and pSyd 8, appear to hybridize weakly to an addition mRNA sequence and one clone, pSyd 3, hybridizes to a multiple mRNA species (1.9, 1.7, 0.9, and 0.5 kilobases). Furthermore, at least one clone, pSyd 2, appears to be expressed only in ER-positive cells. While its level of expression is stimulated by estrogen approximately 4-fold in all the estrogen and progesterone receptor-positive cell lines tested (T-47D, ZR-75-1, and MCF-7), pSyd 2 levels in the estrogen and progesterone receptor-negative HBL-100 cell line were lower than the corresponding levels in estrogen-stimulated T-47D cells and were unresponsive to estradiol. These results show that we have isolated several estrogen-responsive sequences which will be useful in studying hormone regulation of gene expression and may provide additional markers of hormone responsiveness in breast cancer.

Relationship between flow cytometric parameters, steroid receptors, and menopausal status in breast cancers.

Of 163 human breast cancers examined, 68% contained detectable aneuploid populations, whilst 32% had apparently normal DNA distributions. A slightly higher incidence of aneuploidy was observed in pre-menopausal patients (83%) than in post-menopausal patients (66%). Also, pre-menopausal patients had slightly higher proportions of S-phase or cycling (S + G2 + M) cells. Estradiol receptor negative (ER-) tumours from post-menopausal patients were found to have the lowest incidence of aneuploidy (59%) and ER- tumours from pre-menopausal patients the highest (91%). There were no significant differences in the proportions of cycling nuclei when receptor status and menopausal status were considered. A weak relationship is shown to exist between flow cytometric data and two common prognostic indicators of breast cancer, namely receptor status and menopausal status.

Effects of dihydrotestosterone, 17 beta-estradiol and dexamethasone on mitogen--and allogeneic--induced lymphoblastogenesis.

In contrast to the in vivo action of sex steroids the steroidal hormones dihydrotestosterone (DHT) and estradiol (E2) produced a marked variable response on mitogen-induced lymphoblastogenesis. DHT and E2 at concentrations of 10(-11) mol l-1 incubated with mononuclear cell fraction in the presence of mitogens PHA, Con A and PWM caused inhibition, stimulation or no effect. Separation of T and B cell fractions and incubation with DHT and E2 likewise produced variations in response. However, dexamethasone (DEX) strongly inhibited proliferation of all cell fractions, particularly at the higher steroid concentrations. These results suggest that the reported effects of in vivo administered sex hormones on the immune system might possibly be mediated by a precursor cell of peripheral blood mononuclear cells, and that the in vitro system may not be a reproducible test for determining an individual's immunoendocrine status.