Quantification of prostate specific antigen mRNA levels in circulation after prostatic surgery and endocrine treatment by quantitative reverse transcription-polymerase chain reaction.

Various methods to detect prostatic cells in circulation have given conflicting results. This is probably because qualitative rather than quantitative methods have been used to detect mRNA from prostatic cells. A quantitative method has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) for detection of prostate specific antigen (PSA) mRNA in peripheral blood. A competitive internal mRNA standard was used for quantification of absolute amounts of PSA mRNA. The detection limit of the assay was 7 copies of mRNA, and the highest level of circulating PSA mRNA in 88 control subjects was 25 copies per milliliter of blood. This method was used to study the influence of prostatic surgery and endocrine treatment on prostatic cells in the circulation of 56 patients undergoing biopsy, radical prostatectomy, transurethral resection of the prostate (TURP), orchiectomy, or androgen blockade. Blood samples were drawn before, during and up to 26 weeks after these procedures had been carried out. The highest level of PSA mRNA in controls was 25 copies per milliliter of blood. After RP, TURP or orchiectomy, PSA mRNA levels increased above this level in 27%, 29%, and 25% of the samples, respectively. After prostate biopsy, two out of 15 patients became positive. PSA mRNA levels that were elevated by surgery became undetectable within 1-3 days. No significant correlation was found between PCR positivity and the clinical characteristics of the patients. It is concluded that the level of PSA mRNA in peripheral blood increases after prostatic surgery, indicating temporary dissemination of prostatic cells. However, preoperative levels do not correlate with serum PSA, stage or grade.

Quantitative detection of low-copy-number mRNAs differing at single nucleotide positions.

Accurate analysis of mRNA expression levels of SNPs, highly homologous genes, and splicing variants requires techniques capable of quantifying low-copy-number mRNAs differing at single nucleotide positions. We have used an RT-PCR-based technique based on co-amplification of closely related target mRNA transcripts and assessed the effect of the stochastic distribution of low-copy-number templates on sampling variation when quantifying rare mRNA transcripts. The technique was optimized for maximal sensitivity to enable the analysis of samples containing a subpopulation of target cells and small microdissected samples. We demonstrate that the input level of template molecules is a critical determinant of the achievable assay precision. A minimum of approximately 50 molecules of template is required to discriminate between 2-fold differences in the expression levels of two transcripts. At levels above 1000 molecules of input template, the stochastic effects on sampling variation become negligible.

Expression of luteinising hormone and chorionic gonadotropin beta-subunit messenger-RNA and protein in human peripheral blood leukocytes.

Some pituitary hormones are expressed in leukocytes and are thought to play a role in the regulation of leukocyte function. We studied the expression of the mRNA for the beta-chains of luteinising hormone (LHbeta) and chorionic gonadotropin (CGbeta) and their translation into protein in various leukocyte subsets. Monocytes, granulocytes, B and T-cells from peripheral blood were separated. Lymphocytes were stimulated with various mitogens, prolactin and mixed lymphocyte culture. LHbeta and CGbeta mRNA expression was determined by reverse transcriptase polymerase chain reaction. LH, LHbeta, CG and CGbeta protein were determined in the culture medium by immunofluorometric assays. LHbeta mRNA expression was detected in all cell fractions and cultures and stimulation with prolactin induced LH protein in the culture medium. CGbeta mRNA expression appeared after culture of lymphocytes, but mitogens and prolactin had no clear stimulating effect. The LH expression in leukocytes shown here suggests an autocrine function of this hormone in blood cells.

Tumor-associated trypsin inhibitor in normal and malignant renal tissue and in serum of renal-cell carcinoma patients.

Tumor-associated trypsin inhibitor (TATI) is a 6-kDa peptide, which is identical to the pancreatic-secretory-trypsin inhibitor (PSTI). TATI is produced by several tumors and cancer cell lines, and is used as a serum marker for mucinous ovarian cancer. Elevated serum levels of TATI have also been observed in renal-cell carcinoma (RCC). However, it is unclear whether the increase of serum TATI in this disease is caused by production of TATI by the tumor tissue, by the acute-phase reaction frequently associated with cancer, or by impaired renal function. We examined the expression of TATI in malignant and histologically normal renal tissue by immunohistochemistry, in situ hybridization and reverse-transcriptase-polymerase-chain reaction (RT-PCR). Furthermore, we measured pre-operative serum TATI levels in 21 patients with RCC. Immunohistochemically, TATI was detected in 13 of 20 histologically normal renal-tissue samples, but not in 32 tissue samples from RCC. By RT-PCR, TATI mRNA was detected in all of 10 histologically normal kidneys and in 6 of 11 RCCs, while in situ hybridization analysis gave negative results. Pre-operative serum TATI was elevated in 57% of RCC patients. We also studied expression of TATI mRNA and protein in 7 renal-cancer cell lines, by RT-PCR and immunofluorometric assay respectively: 6 cancer cell lines were positive for TATI mRNA, while 4 of them also produced TATI protein at low levels. These results indicate that TATI is synthesized by the histologically normal renal tissue and by some renal cancers, and suggest that the elevation of serum TATI associated with renal-cell carcinoma may be caused by the release of TATI produced by the tumor.

Detection of messenger RNA for the beta-subunit of chorionic gonadotropin in urinary cells from patients with transitional cell carcinoma of the bladder by reverse transcription-polymerase chain reaction.

We studied whether detection of messenger-RNA (mRNA) for the beta-subunit of chorionic gonadotropin (CGbeta) in urinary cells from bladder cancer patients could be used as a marker of disease activity. Sixty-eight urine samples from patients under follow-up for bladder cancer and 23 samples from patients with other malignancies and non-malignant surgical conditions, as well as 14 samples from healthy controls were analyzed. RNA was isolated from urinary cells collected by centrifugation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect CGbeta mRNA. The results were compared to those obtained by cystoscopy and urinary cytology. For comparison, we determined CG and CGbeta in serum and urine and the core fragment of CGbeta (CGbeta cf) in urine by immunofluorometric assays. CGbeta mRNA was detected in 29 of 68 urine samples from patients with a history of bladder cancer, whereas all 14 samples from healthy controls tested negative. Elevated levels of CGbeta were observed in serum in 18 of 45 bladder cancer patients, but the association with CGbeta mRNA was weak. However, CGbeta mRNA expression in the absence of detectable cancer also occurred in some conditions associated with cellular atypia such as urinary tract infection, instrumentation and certain therapies. There was a highly significant association between histologically verified transitional cell carcinoma of the bladder and CGbeta mRNA in urine (p = 0.0014), implying CGbeta mRNA expression in tumor tissue. We conclude that CGbeta mRNA is a potential new marker for monitoring of bladder cancer. Further studies are needed to evaluate whether it provides independent clinical information.

Cellular localization of c-jun messenger ribonucleic acid and protein and their relation to the proliferation marker Ki-67 in the human endometrium.

We studied cellular c-jun messenger RNA (mRNA) expression in the human endometrium during the menstrual cycle (n = 47) and in human decidua during pregnancy (n = 8), by using digoxigenin-labeled RNA probes in in situ hybridization. The same tissue samples were also analyzed for c-Jun protein and the proliferation marker Ki-67. In the proliferative endometrium, strong expression of c-jun was detected in luminal and glandular epithelium as well as in fibroblast-like stromal cells. During the early luteal phase, strong hybridization signals were identified in both epithelial and stromal compartments, with the strongest hybridization in the stromal cells beneath the epithelium. c-jun mRNA was markedly diminished in luminal and glandular epithelia of mid- and late secretory phase endometria, but it remained unchanged in the stroma. Regardless of the phase of the menstrual cycle, significant hybridization was identified in endothelial cells in the endometrium and myometrium, and a low signal was detected in myometrial muscle cells as well. During early gestation, weak expression of c-jun mRNA was observed in glandular epithelial cells and in decidualized stromal cells. In term pregnancy decidua, only low-level hybridization was detected in a few decidual cells. Nuclear immunostaining of c-Jun was detected in luminal and glandular epithelia and in stroma throughout the menstrual cycle. The location of Ki-67 antigen was temporally related to the c-jun mRNA expression in cycling endometrium and pregnancy decidua. From our data we conclude that 1) c-jun mRNA is differentially expressed in endometrial epithelial and stromal cells; 2) c-jun mRNA is cyclically regulated in the human endometrial epithelium; 3) c-jun mRNA expression is temporally related to epithelial proliferation in the endometrium; and 4) c-Jun protein is present in the human endometrium throughout the menstrual cycle.

The expression of prostate-specific membrane antigen in peripheral blood leukocytes.

PURPOSE: Prostate-specific membrane antigen (PSM) and prostate-specific antigen (PSA) have been used as marker genes for detection of cancer cells in circulation of prostate cancer patients. However, PSA was recently found to be expressed in non-prostate cell lines and normal blood. To evaluate this phenomenon for PSM, we studied its mRNA expression in non-prostatic cells and cell lines, in blood from healthy donors and patients with prostate cancer. MATERIALS AND METHODS: We studied PSM expression by a highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood of 24 patients with cancer of the prostate (CAP) and 13 healthy young male and female donors, in four non-prostatic cell lines and in isolated blood cells. The identity of the RT-PCR product was confirmed by sequencing. Contamination of the samples with cDNA or prostatic RNA was rigorously excluded by subjecting each sample to PCR reaction without RT-enzyme and by RT-PCR with PSA primers, respectively. RESULTS: We found PSM mRNA expression in blood from 18 of 24 CAP patients and 12 of 13 healthy donors and in the leukocyte fraction of normal blood cells. PSM expression could not be detected in erythroblasts, platelets, K-562, U-937, HL-60 or Jurkat cell lines. CONCLUSIONS: Our results indicate that sensitive nested RT-PCR method detects PSM mRNA in the leukocyte fraction of normal blood. This "background" expression is probably caused by a leaky promoter of PSM. We conclude that it is necessary to develop quantitative RT-PCR assays to differentiate PSM mRNA expression derived from circulating cancer cells from background expression in blood cells.

Detection of squamous-cell carcinoma antigen-expressing tumour cells in blood by reverse transcriptase-polymerase chain reaction in cancer of the uterine cervix.

We used a reverse transcriptase-polymerase chain reaction method for squamous-cell carcinoma (SCC) antigen mRNA to detect circulating tumour cells in patients with carcinoma of the uterine cervix. The sensitivity of the method, as determined by cell spiking experiments, was 10 cultured A431 cells among 10(6) white blood cells. Circulating tumour cells were detected in 6 of 15 patients. In our control group of 24 women, SCC antigen mRNA was detected in 2 pregnant women at term. We followed up the patients for 24 months after sampling and evaluated the outcome. Three out of 6 patients positive for SCC antigen mRNA have relapsed. Additionally, 1 patient has developed breast cancer. In the group of 9 patients negative for SCC antigen mRNA there has been 1 relapse and 1 case of progression of disease. These results suggest that detection of SCC antigen mRNA in peripheral blood by RT-PCR could be useful for staging and evaluation of prognosis in epidermoid carcinoma of the uterine cervix.