Quality of life after radical trachelectomy for early-stage cervical cancer: A 5-year prospective evaluation.

OBJECTIVES: To longitudinally assess quality of life (QOL) in women undergoing radical trachelectomy for early-stage cervical cancer. METHODS: We prospectively enrolled patients with stage IA1-IB1 cervical cancer prior to undergoing radical trachelectomy to complete validated QOL instruments. These instruments included the General Health-Related QOL (SF-12), Functional Assessment of Cancer Therapy-Cervix (FACT-Cx), MD Anderson Symptom Inventory (MDASI), Female Sexual Functioning Index (FSFI), and Satisfaction with Decision scale (SWD). Instruments were filled out at baseline, postoperatively at 6weeks, 6months, 1year, and annually thereafter for 4years. RESULTS: Thirty-nine patients enrolled in the study, and 32 patients were evaluable. The scores for FSFI-arousal (p=0.0002), lubrication (p<0.0001), orgasm (p=0.006), pain (p=0.01), satisfaction (p=0.03) and total score (p=0.004) showed a significant decline at 6weeks then returned to baseline levels by 6 months. The scores for FACT-Cx functional well-being (p=0.02) and physical well-being (p<0.0001), SF-12 bodily pain (p<0.0001), physical functioning (p<0.0001), role physical (p<0.0001), role emotional (p=0.03), social functioning (p=0.002), and MDASI total (p=0.04) showed significantly worsened symptoms at 6weeks then returned to baseline by 6months. The scores for FACT-Cx emotional well-being showed significant worsening of symptoms that persisted at 6-weeks (p=0.004), 6months (p=0.007), 1year (p=0.001), 2years (p=0.002), and 4 years (p=0.03). There was no difference in SWD. CONCLUSIONS: Several quality of life assessments decline immediately postoperatively after radical trachelectomy, however, return to baseline thereafter. The long-term emotional impact of this surgery highlights a need for perioperative counseling in these patients.

cis-Dominant mutations which dramatically enhance DUR1,2 gene expression without affecting its normal regulation.

We have isolated three cis-dominant mutations which dramatically enhance DUR1 ,2 gene expression in Saccharomyces cerevisiae. The mutant phenotype, which is expressed both in haploid and MATa/MAT alpha diploid strains, does not appear to be an alteration of the normal control system for this gene because its expression remained fully inducible and sensitive to nitrogen catabolite repression. Instead, we found much higher levels of DUR1 ,2-specific RNA under both uninduced and induced conditions, i.e., the overproduction trait was superimposed on normal regulation of the gene. The mutations seemed to affect gene expression in a unidirectional manner or to be specific for DUR1 ,2 gene expression, because other genes in proximity to the mutations were not affected. We feel that these mutations may alter the chromatin structure in the vicinity of the DUR1 ,2 upstream control sequences or, alternatively, may be Ty insertions which no longer possess the ROAM characteristics reported by others and ourselves.

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

Characterization of the retinol and retinoic acid binding proteins in the human prostate.

A retinol-binding protein has been detected in the cytosol of human prostates with benign hyperplasia. The binding was of high affinity and specific for retinol (Kd = 35 nM), with other retinoids such as trans-retinoic acid, retinal, and the synthetic analogues, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona tetraenoic acid and p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1-propenyl] benzoic acid, showing little or no competition. The retinol binding, which sedimented as a 2S component on sucrose density gradients, was also unaffected by the addition of excess unlabeled steroid hormones. Furthermore, pretreatment of the cytosol proteins with heat and/or trypsin totally abolished the retinol binding. Parallel experiments with trans-retinoic acid suggest that the hyperplastic prostate possesses a second retinoid-binding site which is specific for retinoic acid and distinct from the retinol-binding component. Experiments with serum from patients with benign prostate hyperplasia revealed no binding at the 2S sedimentation position; this suggests that the retinoid-binding proteins were exclusively associated with prostatic tissue and were not therefore derived from serum.

Location of the genes that control induction of the allantoin-degrading enzymes in Saccharomyces cerevisiae.

In an effort to understand the regulation of allantoin degradation in Saccharomyces cerevisiae, we isolated two classes of mutants, each defective in the induction process associated with production of the pathway enzymes. Mutation at one locus (DAL80) results in constitutive expression of the genes involved in allantoin catabolism. Mutation at the second locus (DAL-81) results in the loss of ability to induce these enzymes. This report describes genetic data indicating that the DAL80 and DAL81 loci are situated approximately 13 cM from the centromere on the right arm of chromosome XI and 9 cM proximal to the DAL1 locus on chromosome IX, respectively.

Evidence that human prostatic 5 alpha-reductase is located exclusively in the nucleus.

The 5 alpha-reductase activities in human prostatic nuclei and microsomes were compared. The activities in both subcellular fractions were identical with respect to pH dependence, heat inactivation and Michaelis constants for NADPH and testosterone. Subcellular distribution studies using DNA and nicotinamide mononucleotide adenyl transferase as nuclear markers showed that the amount of 5 alpha-reductase present in the microsomes was directly proportional to the amount of nuclear contamination. These results indicate that human prostatic tissue contains only one form of 5 alpha-reductase, which is located exclusively in the nucleus. This finding has important implications for the mechanism of steroid action in the prostate.

Cytokine modulation of epidermal growth factor receptor expression on bladder cancer cells is not a major contributor to the antitumour activity of cytokines.

Epidermal growth factor is a potential mitogen for many different human tumours. Its effect is mediated via a bispecific receptor (EGFR), the expression of which correlates well with invasive disease. We investigated the modulation of EGFR by cytokines produced following bacillus Calmette Guerin (BCG)-immunotherapy. Our data demonstrate the IFN gamma, TNF alpha and IL-1 alpha can decrease the expression of EGFR on some bladder tumour cell lines. IFN gamma reduced EGFR expression on two of eight cell lines (RT4, SD). However, IL-1 and TNF did not share this activity. When cells were treated with a combination of all three cytokines, EGFR was decreased on three cell lines (RT4, RT112, SD) and furthermore, the change in the receptor expression was even more marked. Treatment with phorbol ester (thereby activating protein kinase C) resulted in rapid disappearance of the receptor from the cell surface. Interestingly, the decrease of EGFR expression did not require protein synthesis. Although the cytokines studied could down modulate EGFR, this only occurred on three out of eight cell lines; therefore, it is unlikely that the suppression of proliferative activity caused by cytokine-induced decrease of EGFR expression is central to the antitumour action of BCG therapy, but in a proportion of tumours this mechanism may be involved.

Levels of circulating intercellular adhesion molecule-1 in patients with metastatic cancer of the prostate and benign prostatic hyperplasia.

Current reports suggest a role for intercellular adhesion molecule-1 (ICAM-1) in the progression of malignancy. The availability of a new antibody makes it possible to measure circulating ICAM-1 (cICAM-1) in human body fluids including serum; this might help in monitoring tumour burden and in providing additional prognostic information. In this study, serum levels of cICAM-1 were measured by an ELISA assay in patients with benign prostatic hyperplasia (BPH; n = 20) and metastatic cancer of the prostate (CaP; n = 25). Serum ICAM-1 concentrations were also measured in a group of healthy men (n = 8). The mean +/- S.E.M. cICAM-1 level for BPH was 339.52 +/- 15.30 ng/ml compared with 263.55 +/- 18.54 ng/ml for CaP. Even though the difference between the two groups was significant (P < 0.005), there was a marked overlap between the individual values in both groups, thus minimising the prognostic value of these measurements in prostate cancer. Endocrine therapy had no notable effect on the serum levels of cICAM-1. The mean +/- S.E.M. cICAM-1 concentrations in serum from a younger group of healthy volunteers was 204.1 +/- 10.38 ng/ml, and this value was significantly lower than that measured in serum from either BPH or CaP. We also undertook some immunohistochemical studies to examine the distribution of ICAM-1 in prostate tissue. We observed focal epithelial cell membrane staining which was exceedingly patchy in both the BPH and cancer specimens. On the basis of these studies, we suggest that cICAM-1 levels do not provide additional information on patients with metastatic CaP.

A case for synchronous reduction of testicular androgen, adrenal androgen and prolactin for the treatment of advanced carcinoma of the prostate.

The present study was undertaken mainly to investigate whether prolactin manipulation combined with maximal androgen blockage improves the effectiveness of treatment in advanced prostatic cancer. The efficacy of oral hydrocortisone as an alternative to commercial anti-androgens in reducing the adrenal androgens, and of bromocriptine in reducing the prolactin level were also examined. A consecutive series of 30 patients with untreated and advanced prostatic cancer were entered into a three-arm prospective randomised trial. 10 patients received subcapsular orchiectomy alone (arm 1), another 10 had subcapsular orchiectomy plus flutamide (arm 2), and the remaining 10 had subcapsular orchiectomy plus oral hydrocortisone and bromocriptine (arm 3). Clinical and biochemical parameters, including trans-rectal ultrasound-determined prostatic volumes, hormonal profiles and radionuclide bone scan were evaluated at regular intervals. At 12 months, serum testosterone was reduced by more than 90% in all arms, however, maximum suppression of androstenedione, prolactin, and reduction of prostatic volumes were only observed in arm 3; this was reflected by the significant improvement in clinical response in arm 3 compared with other arms. This study suggests that a combined maximal suppression of androgens and prolactin offers a significant improvement in response over conventional treatments without prolactin suppression in the treatment of advanced prostatic cancer. Importantly, a better clinical outcome in arm 3 was still apparent at the end of 36 months.

The natural history of renal carcinoma.

In summary, some evidence indicating an increase in incidence of renal parenchymal malignancy has been presented. The nature of renal adenoma and its relationship to renal carcinoma remain uncertain, but it is possible that improved computerized tomography will allow in vivo identification of these lesions and initiate a long-term study to provide some clear data on their natural history. The heterogeneity of clinical presentation of this disease has been reviewed and the paraneoplastic syndromes and their importance summarized. Careful clinical and postmortem studies of disease spread, especially lymphatic spread, have been shown to provide useful information to the debate on the role of lymphadenectomy. Many of the unusual aspects of the natural history have been interpreted in terms of the hosts immune response and some data on the complexity and specificity of the host tumor interaction presented. In conclusion, an understanding of the natural history of renal carcinoma forms an important background on which to base clinical management and identifies areas worthy of further investigation in this curious tumor.

5alpha-dihydrotestosterone stimulation of human prostate in organ culture.

Explants of human benign hypertrophied prostate were cultured for 8 days. The effect of 5alpha-dihydrotestosterone on the morphology, DNA and glucose utilization of 14 cases was investigated. In four cases proliferation was clearly more extensive in the dihydrotestosterone-treated explants than in the controls. In six explants, there were no morphological differences between those cultured with or without dihydrotestosterone and in half of these, proliferation was present. Four explants were either necrotic or did not contain epithelial cells. The mean DNA content of the treated explants was higher than that of the controls (P smaller than 0.01). There was no significant difference in the rate of glucose utilization by the explants treated with dihydrotestosterone, and by the controls.

Nuclear retinoic acid binding protein in human prostate adenomas.

A retinoic acid binding protein has been detected in salt extracts of nuclei obtained from human prostate adenoma. The binding was characterized by competition experiments, temperature/time studies and saturation analysis. Substantial binding was only observed after sonication of nuclei and charcoal-pretreatment of a salt extract. The binding of radiolabelled all-trans-retinoic acid was displaced by all-trans-retinoic acid, retinol and to a lesser extent retinal and two synthetic retinoids, RO 10-1670 and RO 13-7410. Testosterone and dihydrotestosterone, at a 100-fold excess, had little effect on the binding. The association between retinoic acid and nuclear protein was both temperature and time dependent. At 37 degrees C, equilibrium was rapidly reached (30 min) whereas at 4 and 25 degrees C, ligand binding occurred at a slower rate. Saturation analysis performed under steady-state conditions yielded a dissociation constant of 15 +/- 2 nmol/l. Metabolism studies failed to show conversion of either radiolabelled all-trans-retinol or [3H]retinoic acid in vitro; these data suggest that both acid and alcohol forms of vitamin A are recognized by the extracted nuclear protein. The effect of three enzyme inhibitors on [3H]retinoic acid binding was studied. Binding was unaltered in the presence of aprotinin and phenylmethylsulphonyl fluoride but sodium molybdate (10 mmol/l) increased binding by 18%. The presence of a specific retinoid binding protein in prostate nuclei suggests that retinoids may play some role in the function of the gland.

Characterization of the prolactin receptor in human prostate.

The uptake of 125I-labelled human prolactin by subcellular fractions obtained from the human hyperplastic prostate was investigated and the specific binding sites were characterized. The binding was both time- and temperature-dependent with maximum specific uptake achieved after incubation for 20 h at 4 degrees C (dissociation constant = 2.4 x 10(-10) mol/l). The present studies also indicate that the bulk of the specific prolactin binding was confined to the 105 000 and 15 000 g membrane fractions whilst the cytosol and nuclear pellet exhibited a lower capacity for the peptide hormone. Attempts to optimize the binding revealed that pretreatment of the subcellular fractions with 4 mol MgCl2/l quadrupled the binding sites; a similar effect was produced after pretreatment with dextran-coated charcoal, but the changes were less pronounced. Furthermore, our studies suggest that the binding was specific for the human prolactin, with other hormones such as ovine prolactin, human LH, human FSH and human GH showing little or no competition. The addition of magnesium and copper ions to the incubation medium also markedly increased the specific binding, whereas calcium, manganese and EDTA inhibited the prolactin uptake. Freezing and storage of tissue at -70 degrees C did not greatly affect the binding sites. The results suggest that we are clearly dealing with a specific prolactin-binding protein.

Interaction between prolactin and zinc in the human prostate gland.

The interaction between prolactin and zinc was examined in vitro in the human prostate gland. The results indicated that prolactin did not modulate the acute uptake of zinc into benign prostatic hypertrophy tissue whereas zinc, in contrast, increased the uptake of prolactin into the prostate gland. Our study further showed that the augmented uptake of prolactin by zinc was partly due to an increase in the non-specific binding properties of the peptide hormone. We were also able to demonstrate that the specific binding of 125I-labelled human prolactin to the receptor was reduced in the presence of zinc by a competitive mechanism.

Androgen metabolism in the epithelial and stromal components of the human hyperplastic prostate.

The reduced and oxidized metabolites of testosterone and dihydrotestosterone were measured in the stromal and epithelial components of 23 human hyperplastic prostates. Our studies indicate differences in the hormonal metabolic patterns of the stroma and epithelium of the resected specimens when compared with tissues obtained retropubically. Testosterone 5 alpha-reductase was evenly distributed between the two components of the specimens obtained retropubically whereas the 3 alpha (beta)-hydroxysteroid dehydrogenase was predominantly located in the stroma. The measurements on the resected specimens suggest, on the other hand, that the bulk of the 5 alpha reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase activities were confined to the stroma although these activities were considerably lower than those measured in the corresponding components of the retropublically obtained specimens. The conversion of testosterone to androstenedione was negligible in all the samples analysed. We therefore conclude that the stroma is the main site for the transformation of dihydrotestosterone to the androstanediol epimers and that the asymmetric distribution of the 3 alpha (beta)-hydroxysteroid dehydrogenase may be instrumental in the development of hyperplasia in the prostate gland. Furthermore, the results of this study indicate that electroresection impairs the enzymatic activities of the tissue.

Localization of epidermal growth factor receptors in the human prostate by biochemical and immunocytochemical methods.

The receptor for epidermal growth factor (EGF) was characterized in the particulate fraction from human benign prostatic hyperplasia (BPH) and was present in 85% of tissues analysed. The uptake of 125I-labelled EGF by BPH was dependent on both time and temperature, with maximum specific uptake achieved after incubation for 90 min at 37 degrees C. Binding characteristics revealed two classes of binding sites of higher (mean dissociation constant (Kd) +/- S.D. = 0.8 +/- 0.2 nmol/l) and lower (Kd = 7.6 +/- 2.8 nmol/l) affinities. Competition studies demonstrated the specificity of the receptor assay since the binding of labelled EGF was abolished with excess unlabelled EGF but not with excess unlabelled human GH, human insulin, venom nerve growth factor, human FSH, human LH and human prolactin. There was a complex biphasic relationship between specific binding and protein concentration in the range 0.1-8 mg/ml. Subcellular fractionation of BPH homogenates demonstrated that the bulk of the specific binding was confined to the 800 g (crude heavy pellet) and 15,000 g (mitochondrial pellet) fractions. The 105,000 g (microsomal pellet) and the 105,000 g (cytosol fraction) exhibited low and variable binding capacities for the growth factor. The presence of EGF receptor was also confirmed by immunocytochemical staining of frozen sections from BPH using monoclonal antibody specific for EGF receptors. A positive correlation between 125I-labelled EGF binding and the intensity of staining was found. The presence of a specific EGF-binding receptor protein in human BPH tissues suggests that EGF may play a role in the pathogenesis of human BPH.

Identification of an androgen receptor in the cytosol of the female Mastomys prostate.

An in vivo and in vitro study was carried out on the prostate from the female Praomys (Mastomys) natalensis to identify and characterize the binding of androgens within the cytoplasm. The labelled cytosol was prepared and subjected to gel exclusion chromatography and density gradient centrifugation. A macromolecular protein associated with the radioactivity was isolated on Sephadex G-200. Subsequent analysis of the steroid receptor complex showed that the major part of the radioactive steroid (64 percent) was dihydrotestosterone. This binding was inhibited by unlabelled testosterone and could not be demonstrated in liver cytosol. Characterization of this dihydrotestosterone receptor complex revealed a sedimentation coefficient of 4.6 s in the presence of a high salt solution (0.4 M KCl). The complex aggregated in the absence of 0.4 M KCl and sedimented preferentially from 5.6-7.4 s together with polydisperse aggregates of higher sedimentation coefficients. The use of this animal as an experimental model for hormonal studies on the prostate is suggested.

Bacillus-calmette-guerin (bcg) organisms directly alter the growth of bladder-tumor cells.

Studies in recent years have shown various effects of bacillus Calmette Guerin organisms on the human immune system. In the present study, the direct effects of bacillus Calmette Guerin (BCG) (as used for the clinical management of superficial bladder cancer) on bladder tumour cells were investigated. Using a proliferation assay, changes in the growth rates of tumour cells were studied following direct exposure to BCG. The effects of variations in the BCG dose, and in the viability of BCG organisms were investigated and our initial observations concerning the antiproliferative effects of interferon-garnma were extended. The main finding of these studies is the direct immuno-modulatory effects of BCG organisms on the proliferative capacity of human tumour cells. Previously these alterations in the growth rate of bladder cancer cells, which are observed following patient therapy, were attributed to the production of various cytokines. However, after exposure to BCG the growth of tumour cell lines was suppressed in a dose and time dependent manner. Furthermore, both viable and nonviable bacilli can exert this action although heat killed BCG may be less effective in doing so. In concordance with our earlier study, interferon-gamma exerted marked antiproliferative effects against eight tumour cell lines. Furthermore, a 12 hour pulse of cytokine was sufficient to suppress the growth of tumour cells. This is an important finding as cytokine is not detected in patient's urine later than 12 hours after immunotherapy. No consistent pattern of growth altering effect was observed with any of the other cytokines tested (IL-1-alpha, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, GM-CSF). Our study suggests that BCG organisms per se may exert direct effects upon tumour cells in vivo and thus ease the load on the immune responses leading to the eventual eradication of tumour.