Looking at tardigrades in a new light: using epifluorescence to interpret structure.

The use of epifluorescence microscopy coupled with ultraviolet (UV) autofluorescence is suggested as a means to view and interpret tardigrade structures. Endogenous fluorochromes are a known component of tardigrade cuticle, claws and bucco-pharyngeal apparatus. By imaging the autofluorescence from tardigrades, it is possible to document these structures in detail, including the subdivisions and boundaries of echiniscid (heterotardigrade) plates and the nature and spatial relationships of the texture (pores, granules, papillae and tubercles) on the various plates. This allows the determination of taxonomic features not easily seen with other microscopic techniques.

Sequential changes in gene expression profiles in breast cancers during treatment with the aromatase inhibitor, letrozole.

The study aim was to identify early (within 14 days) and late changes (by 3 months) in breast cancer gene expression profiles associated with neoadjuvant therapy with letrozole. RNA from sequential tumour biopsies in 54 patients was analyzed on microarrays; changes were determined by frequency, magnitude and significance analyses. Substantially more genes were changed at 3 months (1503) than at 14 days (237). Early changed genes were associated with cell cycle (downregulation), blood vessel development and extracellular matrix (upregulation); late changes included 'cellular metabolic process', 'generation of precursor metabolites and energy' (decreased) and 'cell adhesion' 'biological adhesion' (increased). A striking difference between the early and late changes was the general location of downregulated genes-nuclear structures at 14 days and mitochondria after 3 months. These changes in gene expression profiles provide a new and important database by which to understand molecular mechanisms of letrozole in breast cancers.

Nuclear receptor co-activators and HER-2/neu are upregulated in breast cancer patients during neo-adjuvant treatment with aromatase inhibitors.

BACKGROUND: Acquired resistance to endocrine therapy in breast cancer is poorly understood. Characterisation of the molecular response to aromatase inhibitors in breast cancer tissue may provide important information regarding development of oestrogen hypersensitivity. METHODS: We examined the expression levels of nuclear receptor co-regulators, the orphan nuclear receptor liver receptor homologue-1 and HER-2/neu growth factor receptor using real-time RT-PCR before and after 13-16 weeks of primary medical treatment with the aromatase inhibitors anastrozole or letrozole. RESULTS: mRNA expression of the steroid receptor co-activator 1 (SRC-1) and peroxisome-proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) was correlated (P=0.002), and both co-activators increased during treatment in the patient group as a whole (P=0.008 and P=0.032, respectively), as well as in the subgroup of patients achieving an objective treatment response (P=0.002 and P=0.006). Although we recorded no significant change in SRC-3/amplified in breast cancer 1 level, the expression correlated positively to the change of SRC-1 (P=0.002). Notably, we recorded an increase in HER-2/neu levels during therapy in the total patient group (18 out of 26; P=0.016), but in particular among responders (15 out of 21; P=0.008). CONCLUSION: Our results show an upregulation of co-activator mRNA and HER-2/neu during treatment with aromatase inhibitors. These mechanisms may represent an early adaption of the breast cancer cells to oestrogen deprivation in vivo.

Aromatase and the breast: regulation and clinical aspects.

The aromatase enzyme is unique to the pathway of oestrogen biosynthesis and converts androgen precursors into oestrogens, major stimulatory factors for breast cancer proliferation. Although there is only a single gene for aromatase and a single protein for the enzyme, transcriptional control is complex using different promoters which are in part tissue-specific. These generate different mRNA transcripts that vary in the presence/absence of individual untranslated exon 1s. In breast cancers, species vary between individual tumours, types I.3 and I.4 being the major species in some tumours but type II predominates in the majority. Since the type II promoter is regulated by prostaglandins/cyclic AMP, agents signalling through these systems seem largely responsible for local regulation of intratumoural oestrogen biosynthesis. Autocrine production of these factors would account for the high activity in breast cancers and paracrine secretion for the raised activity in breast fat associated with the local presence of cancer. Given the central role of oestrogen in normal development and pathological processes, there has been great interest in controlling aromatase activity by the use of specific inhibitors. Clinically, this is particularly evident in the management of postmenopausal women with breast cancer.

Aromatase inhibitors and breast cancer.

Oestrogens are involved in risk to, and progression of, breast cancer. Drugs that inhibit the production of oestrogens (aromatase inhibitors, AIs), are therefore extremely attractive strategies both to prevent the disease and to treat established tumours. AIs now have a central role in the treatment of established breast cancer and are being considered for prevention. Third generation agents have been derived from rational drug design. They are able to block oestrogen production and reduce oestrogen levels to a degree that has not been observed previously and without affecting levels of other hormones. Such selective reduction of endogenous oestrogen provides targeted therapy for hormone-dependent breast cancer. This has led to improved clinical benefits in patients with these tumours. Anastrozole, letrozole and exemestane all have impressive antitumour effects in postmenopausal women with breast cancer and they are at least as beneficial as or better than other established endocrine agents when used to treat hormone-sensitive cancer in the advanced setting or as an adjuvant to surgery in earlier stages of the disease; ongoing trials are exploring the use of AIs in the preventative setting. Third generation inhibitors are well tolerated, having no greater side effects than might be expected from oestrogen suppression. Important differences in endocrinological and molecular effects exist between AIs and SERMs. These have implications for the preferred drug sequence and setting in which AIs are used. Since the major obstacle to more widespread use is primary/acquired resistance, discovery of the mechanisms by which resistance occurs offers hope for the future. More detailed study of AIs will yield important information about the involvement of oestrogen on the development and progression of breast cancer. Consequently AIs offer major clinical benefits to patients with breast cancer and the promise of relatively nontoxic intervention in women at high risk of the disease.

Androgen conjugates in human breast cyst fluids.

Dehydroepiandrosterone sulfate (DHAS) has been measured by radioimmunoassay in 100 breast cyst fluids obtained from 82 women. Values ranged from 1.5 to 1,155 microM with a median of 140 microM. These concentrations are in excess of those for plasma but are comparable or less than values for breast secretions obtained by nipple aspiration. Levels of DHAS in cyst fluid were not significantly affected by age, menopausal status, or parity of the subject or by the volume of cyst fluid obtained. In patients with multiple cysts, DHAS values from cysts aspirated from the same breast on the same data were relatively comparable, but wide variations were frequently observed between cysts aspirated on different occasions from the same breast and between cysts from different breasts of the same patient, whether sampled simultaneously or sequentially. Such variability must complicate comparative studies among women.

Hyperprolactinemia and steroid metabolism by rat mammary adenocarcinomas.

The metabolism of both testosterone and dehydroepiandrosterone by 10 adenocarcinomas induced and grown in the presence of high prolactin (plasma prolactin greater than 220 ng/ml) was compared with the metabolism of 10 adenocarcinomas induced and grown in the presence of normal levels (plasma prolactin less than 60 ng/ml). The tumors associated with high prolactin significantly metabolized more testosterone to both 5alpha-dihydrotestosterone and 5alpha-androstanediol. The metabolism of dehydroepiandrosterone was similar in both groups of tumors. It is concluded that prolactin may differentially influence the metabolism of C19 steroids by rat mammary adenocarcinomas.

Significance of aromatase activity in human breast cancer.

The significance of in vitro aromatization of [7 alpha-3H]testosterone to estradiol by human breast cancer has been investigated by correlation with (a) estrogen receptor activity and (b) clinical response to endocrine therapy. Evidence for estradiol synthesis was obtained in 66 and estrogen receptor activity in 81 of 110 tumors. Whereas most estrogen receptor-positive tumors synthesized estradiol, the majority of the receptor-negative tumors did not. This tendency for aromatization to be associated with estrogen receptor-positive tumors was statistically significant (p less than 0.005). Mean level of receptor was also significantly higher in tumors with aromatization than in tumors without estradiol synthesis (p less than 0.001). Forty patients with advanced breast cancer have been treated by endocrine therapy. There was a significant trend for tumors with aromatization to be associated with response to treatment (p less than 0.05), but the correlation was not absolute and may simply reflect the association between aromatase activity and estrogen receptors. Within the small subgroup of patients treated with aminoglutethimide or adrenalectomy, tumors with high aromatase activity responded whereas those without aromatization did not. Tumor estrogen biosynthesis may therefore be of clinical significance in selecting patients for treatments which remove sources of precursor for aromatization of inhibit aromatase activity itself.

Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer.

Expression of the cellular ras Mr 21,000 protein (p21) has been measured in tumors from breast cancer patients who at time of presentation had no evidence of metastatic disease. Western blotting analysis revealed that 37 of 54 (69%) tumors contained p21 levels 2- to 10-fold greater than those of control breast tissues. An excessive increase of p21 (5- to 10-fold over the control value) occurred more frequently in tumors of T3 and T4 stages [15 of 25 (60%)] than in tumors at T2 stage [6 of 29 (21%)], suggesting a correlation between advancement of disease and high p21 levels. p21 levels were positively related to the involvement of axillary lymph nodes at the time of primary treatment. As no correlations were detected between p21 levels and a gross pathological parameter, tumor grade, it is possible that p21 levels may reflect the degree of cellular malignancy. This is supported by data on tumor recurrence; 13 of 16 patients (81%) with tumors expressing low p21 levels were disease free for greater than or equal to 4 years after primary treatment, whereas only 5 of 9 patients (56%) with high p21 tumors remained disease free. These results suggested that a quantitative enhancement of p21 oncogene protein is associated with both the progression and prognosis of breast cancer.

Characterization of the aryl hydrocarbon receptor and aryl hydrocarbon responsiveness in human ovarian carcinoma cell lines.

The human ovarian carcinoma cell lines PE01, PE04, and PE06 express the estrogen receptor and studies with the PE04 cells have shown that tamoxifen inhibits 17 beta-estradiol-induced proliferation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a broad spectrum antiestrogen which works through the aryl hydrocarbon receptor. Incubation of the three cell lines with [3H]TCDD followed by isolation of nuclear extracts showed that the PE01, PE04, and PE06 cells express the aryl hydrocarbon receptor (23 to 87 fmol/mg protein) which exhibits sedimentation properties (7.5 to 7.9 S) on sucrose gradients similar to that observed in other mammalian species. Aryl hydrocarbon responsiveness was determined by the induction of P4501A1 mRNA levels and ethoxyresorufin O-deethylase activity by TCDD. Induction of both parameters was observed only in the PE04 cells. Gel mobility shift assays with a consensus dioxin-responsive element (DRE, 26-mer) showed that after incubation of the nuclear extracts from the 3 cell lines with 32P-DRE a retarded band formed only with nuclear receptor complex from PE04 cells. 17 beta-Estradiol stimulated proliferation of the PE04 and PE06 but not the PE01 cells; 1 nM TCDD alone either did not affect or inhibited the growth of these cells and 1 nM TCDD significantly inhibited the 17 beta-estradiol-induced proliferation of the PE04 and PE06 cells. Treatment of the PE04 cells with 1 nM 17 beta-estradiol resulted in a time-dependent enhanced secretion of the M(r) 52,000 protein (procathepsin D) and, after 48 h, a 51% increase in the secretion of this protein was observed. Cotreatment of the PE04 cells with 0.1 or 1.0 nM TCDD completely inhibited the 17 beta-estradiol-induced secretion of the M(r) 52,000 protein. These data show that TCDD exhibits antiestrogenic activity in estrogen receptor-positive ovarian carcinoma cell lines; however, in the PE06 cells, there was no correlation between the effects of TCDD on the induction of CYP1A1 gene expression and the results of the gel shift assay (i.e., nonresponsiveness) versus the observed antiestrogenic activity.

Growth factor signalling in clinical breast cancer and its impact on response to conventional therapies: the Edinburgh experience.

Neoadjuvant endocrine treatment in which therapy is given while the primary tumour is still in the breast provides a highly useful model system by which to identify mechanisms associated with de novo resistance and signs of early acquired resistance. Most importantly, the model is clinically relevant. It has been confirmed that the absence of tumour oestrogen receptors confers resistance to endocrine therapy. Early changes in tumour cell proliferation following neoadjuvant treatment with the third-generation aromatase inhibitor, letrozole, do not predict accurately for subsequent clinical response. Additionally, changes in proliferation seen at later times can be the consequence of response and may be associated with early resistance. High expression of c-erbB2 does not reduce tumour responses to neoadjuvant treatment with aromatase inhibitors, but is associated with high tumour proliferation before and during treatment. It remains to be determined whether these characteristics confer subsequent resistance to treatment and early relapse in the adjuvant setting.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial.

PURPOSE: Expression of ErbB-1 and ErbB-2 (epidermal growth factor receptor and HER2/neu) in breast cancer may cause tamoxifen resistance, but not all studies concur. Additionally, the relationship between ErbB-1 and ErbB-2 expression and response to selective aromatase inhibitors is unknown. A neoadjuvant study for primary breast cancer that randomized treatment between letrozole and tamoxifen provided a context within which these issues could be addressed prospectively. PATIENTS AND METHODS: Postmenopausal patients with estrogen- and/or progesterone receptor-positive (ER+ and/or PgR+) primary breast cancer ineligible for breast-conserving surgery were randomly assigned to 4 months of neoadjuvant letrozole 2.5 mg daily or tamoxifen 20 mg daily in a double-blinded study. Immunohistochemistry (IHC) for ER and PgR was conducted on pretreatment biopsies and assessed by the Allred score. ErbB-1 and ErbB-2 IHC were assessed by intensity and completeness of membranous staining according to published criteria. RESULTS: For study biopsy-confirmed ER+ and/or PgR+ cases that received letrozole, 60% responded and 48% underwent successful breast-conserving surgery. The response to tamoxifen was inferior (41%, P =.004), and fewer patients underwent breast conservation (36%, P =.036). Differences in response rates between letrozole and tamoxifen were most marked for tumors that were positive for ErbB-1 and/or ErbB-2 and ER (88% v 21%, P =.0004). CONCLUSION: ER+, ErbB-1+, and/or ErbB-2+ primary breast cancer responded well to letrozole, but responses to tamoxifen were infrequent. This suggests that ErbB-1 and ErbB-2 signaling through ER is ligand-dependent and that the growth-promoting effects of these receptor tyrosine kinases on ER+ breast cancer can be inhibited by potent estrogen deprivation therapy.

Aromatase expression in ovarian epithelial cancers.

Our study focused on aromatase cytochrome P450 (CYP19) expression in ovarian epithelial normal and cancer cells and tissues. Aromatase mRNA expression was analyzed by real-time PCR in ovarian epithelial cancer cell lines, in human ovarian surface epithelial (HOSE) cell primary cultures, and in ovarian tissue specimens (n=94), including normal ovaries, ovarian cysts and cancers. Aromatase mRNA was found to be expressed in HOSE cells, in BG1, PEO4 and PEO14, but not in SKOV3 and NIH:OVCAR-3 ovarian cancer cell lines. Correlation analysis of aromatase expression was performed according to clinical, histological and biological parameters. Aromatase expression in ovarian tissue specimens was higher in normal ovaries and cysts than in cancers (P<0.0001). Using laser capture microdissection in normal postmenopausal ovaries, aromatase was found to be predominantly expressed in epithelial cells as compared to stromal component. Using immunohistochemistry (IHC), aromatase was also detected in the epithelium component. There was an inverse correlation between aromatase and ERalpha expression in ovarian tissues (P<0.001, r=-0.34). In the cancer group, no significant differences in aromatase expression were observed according to tumor histotype, grade, stage and survival. Aromatase activity was evaluated in ovarian epithelial cancer (OEC) cell lines by the tritiated water assay and the effects of third-generation aromatase inhibitors (AIs) on aromatase activity and growth were studied. Letrozole and exemestane were able to completely inhibit aromatase activity in BG1 and PEO14 cell lines. Interestingly, both AI showed an antiproliferative effect on the estrogen responsive BG1 cell line co-expressing aromatase and ERalpha. Aromatase expression was found in ovarian epithelial normal tissues and in some ovarian epithelial cancer cells and tissues. This finding raises the possibility that some tumors may respond to estrogen and provides a basis for ascertaining an antimitogenic effect of AI in a subgroup of ovarian epithelial cancers.

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer.

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.

The effects of neoadjuvant anastrozole (Arimidex) on tumor volume in postmenopausal women with breast cancer: a randomized, double-blind, single-center study.

Anastrozole, an orally active, nonsteroidal aromatase inhibitor, was evaluated in a randomized, double-blind, single-center study to determine its efficacy as neoadjuvant therapy in postmenopausal women with newly diagnosed, estrogen receptor-rich, locally advanced or large (>3 cm), operable breast cancers. Twenty-four eligible patients were recruited into the study and received either 1 mg (n = 12) or 10 mg (n = 12) of anastrozole daily over a 3-month period. Tumor volumes were estimated clinically, by using caliper measurements and ultrasound (at baseline and after 1, 2, and 3 months' treatment) and by mammography (at baseline and after 3 months). Tumor volume was also measured in surgical specimens. Twenty-one patients were classified as T2, two patients as T3, and one patient as T4B at baseline. Three patients had clinical evidence of lymph node involvement. When considering the difference between the volume as measured by each assessment and the actual pathological volume, the interquartile range and the difference between the maximum and minimum values were smaller for ultrasound when compared with those measured with calipers and mammography. Therefore, of the three clinical assessments of tumor volume used in this study, the data suggest that ultrasound may be the most accurate. The median reductions in tumor volumes as measured by ultrasound for those patients with a measurable 12-week assessment were 80.5 and 69.6% for anastrozole (1 and 10 mg, respectively) after 12 weeks of treatment and 75.5% when both doses were grouped together. Moreover, of these patients, 11 of 12 given 1 mg and 7 of 11 given 10 mg of anastrozole were found on ultrasound to have a >50% reduction in tumor volume after 12 weeks of treatment. Of the 17 patients who would have required a mastectomy at initiation of treatment, 15 were suitable for breast conservation after anastrozole treatment. These results suggest that anastrozole is highly effective as neoadjuvant therapy in postmenopausal women with estrogen receptor-rich, large, operable breast cancer. Future studies comparing anastrozole with tamoxifen as a neoadjuvant treatment should be considered.

Changes in messenger RNA expression of protein kinase A regulatory subunit ialpha in breast cancer patients treated with tamoxifen.

RNA for protein kinase A regulatory subunit Ialpha (RIalpha) has been measured in tumors from 32 breast cancer patients before and during primary treatment with tamoxifen. Values in pretreatment specimens were significantly higher in tumors subsequently responding to treatment as compared with those not (P = 0.004 by Mann-Whitney U test). Furthermore, whereas levels fell with treatment in 16 of the 24 responding tumors, they did not in any of the 8 tamoxifen-resistant tumors (and indeed rose in 6 cases). These results suggest that measurement of RIalpha mRNA may help in identifying endocrine-dependent breast cancers and provide further evidence of the involvement of the protein kinase A system in response and resistance to tamoxifen treatment.

Cyclic adenosine 3',5'-monophosphate-binding proteins in human ovarian cancer: correlations with clinicopathological features.

The regulatory subunits of protein kinase A, or cyclic AMP-binding proteins, were measured in a series of 107 human ovarian tumors (89 malignant, 7 borderline, and 11 benign tumors) and related to tumor clinicopathological features and patient survival. Total cyclic AMP-binding protein levels were not significantly different between malignant tumors and either borderline or benign tumors. However, serous tumors showed significantly higher levels of total cyclic AMP-binding proteins than other malignant tumors (P = 0.007). Poorly differentiated tumors also possessed significantly higher levels of binding proteins as compared with well/moderately differentiated tumors (P < 0.01). Retrospective analysis of follow-up data also revealed a significant trend for patients with high tumor cyclic AMP-binding proteins to have poorer survival (P = 0.03). Individual binding proteins were identified by photoaffinity labeling, and the RI (Mr 48,000) protein was expressed as a percentage of total cyclic AMP-binding proteins detected. The percentage of the RI protein was not significantly different among malignant, borderline, or benign pathologies and was not associated with tumor stage, differentiation, or debulk status. The percentage of RI was significantly increased in serous tumors compared to other common epithelial malignancies (P = 0.01). In malignant tumors there was a significant positive correlation between the percentage of the RI protein and total cyclic AMP-binding proteins (P = 0.01). These data indicate that high tumor levels of cyclic AMP-binding proteins are associated with serous histology, poor differentiation, and poor patient survival.

Functionality of the progesterone receptor in ovarian cancer and its regulation by estrogen.

Here, we sought to obtain evidence that the progesterone receptor (PR) may be functional in ovarian cancer and regulated by estrogen. Megestrol acetate inhibited growth of the PR-positive PE04 ovarian carcinoma xenograft but not the PR-negative HOX 60 xenograft. PR concentration was higher in early-stage (I/II) tumors than in advanced-stage (III/IV) tumors (P = 0.007) and in tumors of endometrioid histology compared to other carcinoma subtypes (P = 0.009). Patients with a tumor PR concentration of >40 fmol/mg protein had significantly improved survival over those patients whose tumors contained <40 fmol/mg (P = 0.0007; log-rank). Evidence of PR regulation by estrogen was obtained by endocrine manipulation of the PE04 xenograft. PR content of PE04 xenografts fell from 145 to 7 fmol/mg protein in ovariectomized mice and was 2 fmol/mg in male mice. Administration of 17-beta-estradiol increased PR content to 745 fmol/mg. In primary ovarian carcinomas, PR was significantly associated with ER concentrations (P < 0.0001), suggesting regulation of PR levels by estrogen. This association was present for tumors of endometrioid histology (P < 0.0001) but not for those with serous histology (P = 0.31). These data point to the regulation of PR levels by estrogen in ovarian cancer and to a mediatory role for PR in the inhibition of growth induced by progestin.

Expression of the heat shock protein HSP27 in human ovarian cancer.

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.