Control of cell proliferation by steroids: the role of 17HSDs.

Sex steroid hormone signaling regulates the development, growth, and functioning of the breast and the prostate and plays a role in the development and progression of cancer in these organs. The intracellular concentration of active sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSDs). Changes in the expression patterns of these enzymes may play a pathophysiological role in malignant transformation. We recently analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in about 800 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. Cox multivariate analyses showed that 17HSD type 1, tumor size, and estrogen receptor alpha (ERalpha) had independent prognostic significance. We developed, using a LNCaP prostate cancer cell line, a model to study the malignant transformation of prostate cancer and showed that androgen-sensitive LNCaP cells are transformed into neuroendocrine-like cells when cultured without androgens and, eventually into highly proliferating androgen-independent cells. We conducted Northern hybridizations and microarrays to analyze the gene expression during these processes. Substantial changes in the expressions of steroid metabolizing enzymes occurred during the transformation process. The variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

Enzymes as modulators in malignant transformation.

Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ERalpha had independent prognostic significance. Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

17 beta-hydroxysteroid dehydrogenases--their role in pathophysiology.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) regulate the biological activity of sex steroid hormones in a variety of tissues by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone, and corresponding less active hormones, estrone and androstenedione. Epidemiological and endocrine evidence indicates that estrogens play a role in the etiology of breast cancer, while androgens are involved in mechanisms controlling the growth of normal and malignant prostatic cells. Using LNCaP prostate cancer cell lines, we have developed a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition into more aggressive cells. Our data suggest that substantial changes in androgen and estrogen metabolism occur in the cells, leading to increased production of active estrogens during the process. In breast cancer, the reductive 17HSD type 1 activity is predominant in malignant cells, while the oxidative 17HSD type 2 mainly seems to be present in non-malignant breast epithelial cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach in treating estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered to be estrogen target tissues, such as the gastrointestinal tract.

Sex steroid hormone metabolism and prostate cancer.

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.

Comparison of human prostate specific glandular kallikrein 2 and prostate specific antigen gene expression in prostate with gene amplification and overexpression of prostate specific glandular kallikrein 2 in tumor tissue.

BACKGROUND: There is a need for specific markers that can indicate the different stages of prostate carcinoma. There is ongoing speculation concerning the value of prostate specific glandular kallikrein (hK2) in this regard. METHODS: The expression levels of both hK2 and human prostate specific antigen (hPSA) were compared at the mRNA and protein levels by using in situ hybridization and immunohistochemistry techniques in tissue specimens from patients with benign prostatic hyperplasia and malignant prostate carcinoma. The respective gene copy numbers were analyzed by a competitively differential polymerase chain reaction-based method. RESULTS: In situ hybridization revealed that hK2 was expressed at significantly higher levels in malignant prostate tissue compared with benign prostate tissue (P < 0.0005), whereas hPSA expression levels were the reverse (P = 0.06). In benign tissue, the mean level of hK2 mRNA was 82% of the respective value of hPSA (P < 0.003), whereas, in tumor tissue, the mean hK2 expression level was 21% higher than that of hPSA (P < 0.01). The results at the protein level supported the mRNA findings: hPSA expression was lower in malignant tissues compared with benign tissues (17 of 20 specimens), whereas an increase in hK2 expression was detected in 17 of 19 specimens. The authors report that the hK2 gene (hKLK2) was amplified in prostate carcinoma tissue, whereas the hPSA gene was not. There was a correlation between hPSA and hK2 mRNA levels in both benign tissue (correlation coefficient [r] = 0.735; P < 0.01) and malignant tissue (r = 0.767; P < 0.01). CONCLUSIONS: Gene amplification of hKLK2 may be one of the factors leading to higher expression of hK2 in prostate carcinoma. The correlation between hK2 and hPSA expression levels indicates coordinated expression of the genes in both normal and abnormal prostate gland. The results suggest the potential value of hK2 in the diagnosis of prostate carcinoma through mRNA analyses and gene amplification.

Specific DNA binding and transactivation potential of recombinant, purified Stat5.

The signal transducers and activators of transcriptions (Stats) are central mediators of cytokine responses especially in hematopoietic cells. The detailed molecular mechanisms of Stat activation, particularly the role of post-translational modifications and co-operation with cellular transcription factors are subject to intense investigation. The phosphorylation of a tyrosine residue in the carboxyl terminal domain is a common characteristic for the biologically active state of all known Stats. We studied the biological potential of purified recombinant murine Stat5a and Stat5b. These proteins were expressed in Sf9 insect cells upon infection with Stat5 encoding baculoviruses. We also obtained the tyrosine phosphorylated, activated forms of the Stat5 proteins by expressing the tyrosine kinase Janus kinase2 (Jak) in the same cells through co-infection with a kinase encoding virus. After purification, only the tyrosine phosphorylated form was able to bind specifically in vitro to the Stat5 DNA response element. This activated form of Stat5 is also able to support specific cell free in vitro transcription of a gene with a Stat5 response element in its promoter region. The recombinant purified Stat5 proteins were treated with the tyrosine specific protein phosphatase or with potato acidic phosphatase, which removes phosphate groups from serine and tyrosine residues. Phosphatase treatment resulted in the loss of specific DNA binding ability. This property could be restored by an in vitro reaction with recombinant, purified EGF or PDGF receptor kinases. Tyrosine rephosphorylation in vitro also restored the transactivation potential of Stat5. This modification is, therefore, a sufficient prerequisite for transcriptional induction by Stat5.

Androgen-sensitive human prostate cancer cells, LNCaP, produce both N-terminally mature and truncated prostate-specific antigen isoforms.

To characterize prostate-specific antigen (PSA) produced by cancer cells, different isoforms of PSA secreted by the human prostate cancer cells, LNCaP, were purified. LNCaP-PSA production was induced by synthetic androgen, R1881. LNCaP-PSA was separated into four pools. The molecular mass of LNCaP-PSA isoforms in these pools was 34 kDa under reducing conditions and 29 kDa under nonreducing conditions on SDS/PAGE. pI of LNCaP-PSA isoforms varied from 6.8 to 8.2. Pool A had the highest specific activity, 37 nmol/(min x mg). All the pools formed stable complexes with alpha1-antichymotrypsin and alpha2-macroglobulin. The pools contained 10-60% of N-terminally correctly processed LNCaP-PSA isoforms. According to the molecular modelling, the addition or deletion of two or four N-terminal amino acids could affect the three-dimensional structure and thereby remarkably reduce the enzyme activity of LNCaP-PSA.

Human prostate-specific glandular kallikrein is expressed as an active and an inactive protein.

A polymorphism in the human prostate-specific glandular kallikrein (hKLK2) gene was described by direct sequencing (by PCR) of genomic DNAs isolated from prostatic cancer tissue, benign prostatic hyperplasia tissue, and blood leukocyte specimens. Results showed two forms of human prostate-specific glandular kallikrein protein (hK2), a consequence of a change from C to T at base 792 in the hK2 coding region. Producing the two forms as recombinant proteins in insect cells demonstrated that Arg226-hK2 (CC genotype) is an active protein and Trp226-hK2 (TT genotype) is inactive. Polymorphism studies of 36 patients with prostatic diseases identified only 1 with the TT genotype. The same kind of polymorphism was not detected in the human prostate-specific antigen (hKLK3) gene. Arg226-hK2 possessed only trypsin-like enzyme activity, whereas recombinant human prostate-specific antigen (hPSA) had only chymotrypsin-like activity. Monoclonal and polyclonal antibodies raised against hPSA purified from seminal plasma detected both active and inactive hK2. Thus, because inactive as well as stable hK2 protein may be present, a lack of trypsin-like activity in hPSA standards is not enough to confirm that the materials are free of hK2 contamination.

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale.

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.

Site-directed mutagenesis of prostatic acid phosphatase. Catalytically important aspartic acid 258, substrate specificity, and oligomerization.

At the active site of rat prostatic acid phosphatase (rPAP), residue Asp258 is a suitable candidate to act as an acid/base catalyst during phosphoester hydrolysis. It was changed to Asn, Ser, and Ala by site-directed mutagenesis. All these mutants were inactive, indicating that Asp258 may act as a proton donor in catalysis. Tyr123 and Arg127 residues, located at the entrance of the active site surface in rPAP, are likely to be responsible for the substrate specificity of the enzyme. The corresponding residues in lysosomal acid phosphatase (LAP) are Lys and Gly. In order to clarify the roles of the Tyr123 and Arg127 residues, lysosomal type rPAP mutants (Y123K, R127G and Y123K,R127G) were generated. Sensitivity of Y123K,R127G to tartrate inhibition was similar to that observed in the case of LAP, indicating that these residues might be responsible for differences in substrate specificity between the enzymes of prostatic and lysosomal origin. However, unlike human LAP, the lysosomal type mutants hydrolyzed the suggested PAP-specific substrates, phosphocreatine and phosphocholine, showing that Tyr123 and Arg127 are not the only residues contributing to the substrate specificity of rPAP. The residues Trp106 and His112 appeared to be important in the dimerization of rPAP. Oligomerization mutants (W106E, H112D and W106E,H112D) existed in a monomeric form without catalytic activity or a tartrate binding ability.

Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.

Template-primer activity of 5-(hydroxymethyl)uracil-containing DNA for prokaryotic and eukaryotic DNA and RNA polymerases.

We have utilized Bacillus subtilis phage SPO-1 DNA as a model of irradiated DNA. In this phage, all thymine (Thy) residues are replaced by 5-(hydroxymethyl)uracil (5HmUra), which is a known irradiation-induced derivative of DNA Thy. SPO-1 phage is naturally devoid of other such irradiation-induced DNA lesions. DNase I activated SPO-1 phage DNA served as well as, or even better than, the control DNAs (Bacillus subtilis DNA and calf thymus DNA) as a template-primer for Escherichia coli, Micrococcus luteus, and human HL-60 cell DNA polymerases. Furthermore, the template activity of SPO-1 phage DNA was also superior when transcription with E. coli RNA polymerase was investigated. The results reported here indicated that the replacement of Thy by 5HmUra is not deleterious to template and primer functions during DNA or RNA synthesis.