The mitochondrial DNA constitution shaping T-cell immunity in patients with rectal cancer at high risk of metastatic progression.

PURPOSE: A significant percentage of colorectal cancer patients proceeds to metastatic disease. We hypothesised that mitochondrial DNA (mtDNA) polymorphisms, generated by the high mtDNA mutation rate of energy-demanding clonal immune cell expansions and assessable in peripheral blood, reflect how efficiently systemic immunity impedes metastasis. PATIENTS AND METHODS: We studied 44 rectal cancer patients from a population-based prospective biomarker study, given curative-intent neoadjuvant radiation and radical surgery for high-risk tumour stage and followed for metastatic failure. Blood specimens were sampled at the time of diagnosis and analysed for the full-length mtDNA sequence, composition of immune cell subpopulations and damaged serum mtDNA. RESULTS: Whole blood total mtDNA variant number above the median value for the study cohort, coexisting with an mtDNA non-H haplogroup, was representative for the mtDNA of circulating immune cells and associated with low risk of a metastatic event. Abundant mtDNA variants correlated with proliferating helper T cells and cytotoxic effector T cells in the circulation. Patients without metastatic progression had high relative levels of circulating tumour-targeting effector T cells and, of note, the naïve (LAG-3+) helper T-cell population, with the proportion of LAG-3+ cells inversely correlating with cell-free damaged mtDNA in serum known to cause antagonising inflammation. CONCLUSION: Numerous mtDNA polymorphisms in peripheral blood reflected clonal expansion of circulating helper and cytotoxic T-cell populations in patients without metastatic failure. The statistical associations suggested that patient's constitutional mtDNA manifests the helper T-cell capacity to mount immunity that controls metastatic susceptibility. TRIAL REGISTRATION: ClinicalTrials.gov NCT01816607; registration date: 22 March 2013.

Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer.

BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer. METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F). RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01). CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.

Expression of matrix metalloproteinase-2 in serous borderline ovarian tumors is associated with noninvasive implant formation.

OBJECTIVE: To investigate matrix metalloproteinase (MMP) proteolytic and vascular endothelial growth factor (VEGF) and receptor (VEGFR-1, VEGFR-2) angiogenetic capacity in serous borderline ovarian tumors (S-BOTs) for women with and without noninvasive implants. METHODS: The population was made up of 99 patients with S-BOTs as the primary diagnosis between 1985 and 1995, 44 of whom had noninvasive implants and 55 without implants. MMP-2, MMP-14, the type-2 tissue inhibitor of MMPs (TIMP-2), and VEGF and receptors (VEGFR-1, VEGFR-2) were examined by immunhistochemistry. RESULTS: Strong positive (+++) MMP-2 staining was found more frequently in women with primary S-BOTs and noninvasive implants (76%) than in those without implants (53%; p < 0.05). In contrast, staining for MMP-14 and TIMP-2 was not significantly different in the two groups. Furthermore, expression of MMP-2, MMP-14, and TIMP-2 was similar in primary tumors and in their noninvasive implants. Most tumors in both groups had no VEGF expression (84% in the noninvasive implant group and 82% in the group without implants), while moderate (++) to strong (+++) expression of VEGFR-1 and VEGFR-2 was detected in 79% and 94% of the two tumor groups, with no significant difference between the groups. CONCLUSIONS: Enhanced MMP-2 was seen in primary S-BOT with noninvasive implants. The presence of noninvasive implants was prognostic for disease-free survival.

Biphasic regulation of the messenger ribonucleic acid coding for the estrogen receptor by cyclic adenosine 3':5'-monophosphate in tumor Leydig cells.

In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo cyclic AMP (8-Br-cAMP) and human chorionic gonadotropin in a mouse tumor Leydig cell line (MA-10 cells). When the MA-10 cells were cultured in the presence of the cAMP analogue for varying time periods, a transient increase in the level of ER mRNA was observed. Short time incubation (0-2 h) with 8-Br-cAMP enhanced the expression of ER mRNA (2-fold), whereas longer times of incubation (6 h) had the opposite effect (the level of ER mRNA was reduced by 60-70%). The inhibitory effect of 8-Br-cAMP on ER mRNA was not counteracted by aminoglutethimide, an inhibitor of steroidogenic enzymes, indicating that this effect is not mediated via steroids (progesterone). Treatment of 8-Br-cAMP for 6 h caused a concentration-dependent inhibition of ER mRNA with a half-maximal effect of approximately 150 microM. Increasing concentrations of human chorionic gonadotropin for 6 h was also associated with a biphasic effect on the ER mRNA level. Low concentrations (0.20-0.40 ng/ml) increased ER mRNA in the MA-10 cells whereas the highest concentration (20 ng/ml) caused a suppression of this mRNA. In contrast to the biphasic effects observed for the ER mRNA, the level of the regulatory subunit type II beta of the cAMP-dependent protein kinase (protein kinase A) was enhanced in a concentration-dependent manner by human chorionic gonadotropin. Furthermore, 8-Br-cAMP stimulated the mRNA for regulatory subunit type II beta (10- to 20-fold) by all concentrations examined (50-1000 microM). The observations reported here indicate that the expression of ER mRNA is regulated both by endogenously formed and exogenously added cAMP and that there may exist regulatory loops between the steroid and the cAMP/protein kinase A systems.

Expression of a novel factor in human breast cancer cells with metastatic potential.

Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.

Expression of a novel factor, com1, is regulated by 1,25-dihydroxyvitamin D3 in breast cancer cells.

Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5-8-fold) of com1 mRNA. Furthermore, clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)2D3 (10(-7) M), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.

Oxaliplatin-containing Preoperative Therapy in Locally Advanced Rectal Cancer: Local Response, Toxicity and Long-term Outcome.

AIMS: This non-randomised study was undertaken to examine oxaliplatin as possibly an intensifying component of sequential neoadjuvant therapy in locally advanced rectal cancer for improved local and metastatic outcome. MATERIALS AND METHODS: Ninety-seven patients (57 T2-3 cases, 40 T4 cases) received two cycles of the Nordic FLOX regimen (oxaliplatin 85 mg/m(2) day 1 and bolus 5-fluorouracil 500 mg/m(2) and folinic acid 100 mg days 1 and 2) before long-course chemoradiotherapy with concomitant oxaliplatin and capecitabine, followed by pelvic surgery. Treatment toxicity, local tumour response and long-term outcome were recorded. RESULTS: Good histologic tumour regression was obtained in 72% of patients. Implementing protocol-specific dose adjustments, tolerance was acceptable and 95% of patients received the total prescribed radiation dose. Estimated 5 year progression-free and overall survival were 61% and 83%, respectively. T4 stage was associated with an inferior local response rate, which again was highly associated with impaired long-term outcome. CONCLUSIONS: In this cohort of rectal cancer patients dominated by T4 and advanced T3 cases given sequential oxaliplatin-containing preoperative therapy with acceptable toxicity, high tumour response rates and overall survival were obtained, consistent with both local and systemic effects. However, tumour response and long-term outcome remained inferior for a significant number of T4 cases, suggesting that the T4 entity is biologically heterogeneous with subgroups of patients eligible for further individualisation of therapy.

Expression of a novel factor, com1, in early tumor progression of breast cancer.

Tumor cells and their surrounding microenvironment produce a variety of growth factors and proteolytic enzymes to promote tumor growth and metastasis. We have recently identified a novel factor, termed com1, which is up-regulated in human breast carcinoma cells upon formation of experimental metastatic tumors and assumed to act as a growth-promoting factor in breast cancer. In attempts to explore the biological role of com1 in clinical tumor growth and metastasis, expression of com1 mRNA in primary carcinomas from 81 breast cancer patients and 27 samples of uninvolved adjacent breast tissue from these patients was compared and related to known prognostic parameters and outcome. The levels of com1 mRNA were significantly up-regulated (P < 0.0001) in the tumors compared to the normal breast tissues. Tumor expression of com1 mRNA, however, did not correlate with the mRNA levels for urokinase-type plasminogen activator (uPA), its receptor, or the type 1 inhibitor, which are factors that define a phenotype of tumor aggressiveness when elevated. And whereas the mRNA levels of uPA and the uPA receptor were elevated in tumors from the patients who subsequently had poor outcome, no correlations were observed between tumor com1 mRNA expression and prognosis or histological and biochemical characteristics of the tumors. We therefore assume that com1 may mediate some growth-promoting function early in development of the primary breast carcinoma, but not in later stages of tumorigenesis or metastasis.

High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases.

Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular functions, particularly the contrasting activities of inhibiting tissue-degrading enzymes and promoting cellular growth. In attempts to elucidate which of these functions may prevail in breast cancer, expression of mRNAs for TIMP-1 and TIMP-2 in the primary carcinomas from 34 breast cancer patients was related to known prognostic parameters and the clinical outcome. High levels of TIMP-1 mRNA showed significant correlation with the presence of lymph node metastases (P = 0.0067), development of distant metastases (P = 0.014), and early death of the disease (P = 0.020). Elevated expression of TIMP-2 mRNA was associated with development of distant metastases (P = 0.0055). No correlations, however, were observed between mRNA levels of TIMPs and prognostic factors such as patient age, tumor size, grade of anaplasia, or steroid receptor status; neither were any correlations found between these clinicopathological characteristics and the mRNA expression of the collagenolytic enzymes matrix metalloproteinase-2 and matrix metalloproteinase-9. The present data suggest that high levels of TIMP-1 and TIMP-2 mRNAs in the primary carcinomas are strongly associated with development of metastasis in breast cancer.

Personalized radiotherapy: concepts, biomarkers and trial design.

In the past decade, and pointing onwards to the immediate future, clinical radiotherapy has undergone considerable developments, essentially including technological advances to sculpt radiation delivery, the demonstration of the benefit of adding concomitant cytotoxic agents to radiotherapy for a range of tumour types and, intriguingly, the increasing integration of targeted therapeutics for biological optimization of radiation effects. Recent molecular and imaging insights into radiobiology will provide a unique opportunity for rational patient treatment, enabling the parallel design of next-generation trials that formally examine the therapeutic outcome of adding targeted drugs to radiation, together with the critically important assessment of radiation volume and dose-limiting treatment toxicities. In considering the use of systemic agents with presumed radiosensitizing activity, this may also include the identification of molecular, metabolic and imaging markers of treatment response and tolerability, and will need particular attention on patient eligibility. In addition to providing an overview of clinical biomarker studies relevant for personalized radiotherapy, this communication will highlight principles in addressing clinical evaluation of combined-modality-targeted therapeutics and radiation. The increasing number of translational studies that bridge large-scale omics sciences with quality-assured phenomics end points-given the imperative development of open-source data repositories to allow investigators the access to the complex data sets-will enable radiation oncology to continue to position itself with the highest level of evidence within existing clinical practice.

MRI volumetry for prediction of tumour response to neoadjuvant chemotherapy followed by chemoradiotherapy in locally advanced rectal cancer.

OBJECTIVE: To investigate if MRI-assessed tumour volumetry correlates with histological tumour response to neoadjuvant chemotherapy (NACT) and subsequent chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC). METHODS: Data from 69 prospectively enrolled patients with LARC receiving NACT followed by CRT and radical surgery were analysed. Whole-tumour volumes were contoured in T2 weighted MR images obtained pre-treatment (VPRE), after NACT (VNACT) and after the full course of NACT followed by CRT (VCRT). VPRE, VNACT and tumour volume changes relative to VPRE, ΔVNACT and ΔVCRT were calculated and correlated to histological tumour regression grade (TRG). RESULTS: 61% of good histological responders (TRG 1-2) to NACT followed by CRT were correctly predicted by combining VPRE < 10.5 cm(3), ΔVNACT > -78.2% and VNACT < 3.3 cm(3). The highest accuracy was found for VNACT, with 55.1% sensitivity given 100% specificity. The volume regression after completed NACT and CRT (VCRT) was not significantly different between good and poor responders (TRG 1-2 vs TRG 3-5). CONCLUSION: MRI-assessed small tumour volumes after NACT correlated with good histological tumour response (TRG 1-2) to the completed course of NACT and CRT. Furthermore, by combining tumour volume measurements before, during and after NACT, more good responders were identified. ADVANCES IN KNOWLEDGE: MRI volumetry may be a tool for early identification of good and poor responders to NACT followed by CRT and surgery in LARC in order to aid more individualized, multimodal treatment.

Down-regulation of messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) by phorbol ester requires ongoing RNA synthesis but not protein synthesis. Is hormonal control of ER mRNA degradation mediated by an RNA molecule?

Treatment of MCF-7 cells, a human mammary adenocarcinoma cell line, with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10-7 M) was associated with a time-dependent reduction in the level of estrogen receptor (ER) mRNA (half-life approximately 3 h). In the presence of the RNA synthesis inhibitor actinomycin D [5.0 micrograms/ml (4.0 microM)], half-life of ER mRNA was much longer (approximately 12 h). Furthermore, the TPA-dependent down-regulation of ER mRNA was abolished by actinomycin D. Similar effects were observed with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (150 microM), an inhibitor of RNA polymerase. Inhibition of protein synthesis by cycloheximide (50 microM) or puromycin [50 micrograms/ml (92 microM)] did not alter the steady state level of ER mRNA during a period of 10-12 h. Furthermore, these inhibitors of protein synthesis did not prevent the down-regulation of ER mRNA in the presence of TPA. Our studies show that degradation of ER mRNA by TPA in MCF-7 cells is dependent on ongoing RNA synthesis but not on protein synthesis. This indicates that an RNA molecule with rapid turnover, which does not require translation, might be involved in the TPA-dependent ER mRNA decay.

Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells.

In this report we demonstrate glucocorticoid receptors in seminiferous tubules of the rat testis, and that these receptors are localized in Sertoli cells and peritubular cells. The receptors had high affinity for [3H]dexamethasone (Kd = 0.5 - 1 x 10(-9) M), and similar Kd values were calculated from equilibrium analysis and from rate studies (k1 = 1.5 x 10(6) M-1 min-1 and k-1 = 1.4 x 10(-3) min-1, O C). Binding specificity was typical for glucocorticoid receptors (affinity: dexamethasone greater than corticosterone greater than cortisol approximately R5020 approximately progesterone greater than aldosterone = R1881 greater than 17 beta-estradiol approximately cortisone approximately testosterone greater than 5 alpha-dihydrotestosterone). The concentration of glucocorticoid receptors in rat seminiferous tubules revealed an age-dependent decrease, coinciding with the increase in the number of germ cells. Glucocorticoid receptor levels were higher in Sertoli cells from immature rats than in cells from adult rats. Cultured peritubular cells from immature rats contained levels of glucocorticoid receptors similar to cultured Sertoli cells from rats of the same age. With a nick-translated human glucocorticoid receptor complementary DNA probe, a messenger RNA (mRNA) species of approximately 7 kilobase was clearly detected in both Sertoli cells and peritubular cells. In peritubular cells, a smaller mRNA species (5 kilobase) was also clearly detectable. In mRNA from whole testis tissue, a similar developmental pattern as for dexamethasone binding was found. Dexamethasone caused a concentration-dependent stimulation of mRNA levels for androgen binding protein and for the cAMP-dependent protein kinase regulatory subunit type II beta in cultured immature rat Sertoli cells. On the other hand, mRNA levels for glucocorticoid receptor decreased, whereas mRNA levels for beta-actin remained constant. This report documents for the first time the presence of glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells, and is also the first demonstration of glucocorticoid receptors in peritubular cells of the rat testis.

1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor number and increasing the level of the AC inhibitory G-protein Gi-2 alpha in FRTL-5 cells.

Down-regulation of messenger ribonucleic acid and protein levels for estrogen receptors by phorbol ester and calcium in MCF-7 cells.

Treatment of MCF-7 cells, a human mammary carcinoma cell line, with phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] or calcium ionophore (A23187) was associated with striking effects on levels of estrogen receptor (ER) mRNA, specific binding of 17 beta-[3H]estradiol [( 3H]E2), and immunoreactive ER. TPA (10(-7) M) caused a time-dependent reduction of ER mRNA which was below the level of detection after 9 h. Similar effects of TPA appeared at levels of specific binding of [3H]E2 as well as immunoreactive ER. In contrast, TPA induced an increase in mRNA for beta-actin. Incubation of MCF-7 cells with increasing concentrations of TPA (10(-11)-10(-7) M) was associated with biphasic effects on ER mRNA and proteins. Levels of immunoreactive progesterone receptors (PR) were induced by E2 (10(-9) M) in a time-dependent manner. In the presence of TPA (10(-7) M), where ER levels were suppressed, no induction of PR was observed. Removal of TPA (10(-7) M) after 10 h (ER mRNA) or 22 h (ER proteins) of treatment was associated with a continued suppression of both mRNA and protein levels during the entire incubation period (48 h). Treatment with A23187 (2 x 10(-7) M) also caused a time-dependent down-regulation of levels of ER mRNA and proteins. These effects occurred somewhat more slowly than those of TPA. Levels of beta-actin mRNA were not changed by this treatment. These results indicate that changes in estrogen sensitivity are mediated by calcium-dependent protein kinases in human mammary carcinoma MCF-7 cells.

Autologous down-regulation of messenger ribonucleic acid and protein levels for estrogen receptors in MCF-7 cells: an inverse correlation to progesterone receptor levels.

In the present study we have examined the effects of estradiol on mRNA levels for estrogen (ER) and progesterone receptors (PR) in the estrogen-dependent mammary carcinoma MCF-7 cell line. The changes in ER immunoactivity and specific binding of [3H]R5020 were also assessed. Estradiol (10(-7) M) caused a transient and time-dependent reduction of the level of mRNA for ER, with a maximal effect (30-40% of control; n = 3) after 72 h. This was associated with a similar decrease in ER immunoactivity. Further treatment (96 and 120 h) revealed a return of ER mRNA to control values, whereas the ER immunoactivity remained depressed. The effect on the mRNA level for PR gave almost the inverse curve. Initially (24-72 h), we observed a pronounced increase in this mRNA, with a maximal effect (6-7 times the control value; n = 3) after 72 h. Treatment beyond 72 h was associated with a gradual return of mRNA for PR toward the control level. The variation in specific binding of [3H]R5020 revealed similar changes, except that changes in specific receptor binding were delayed 24 h compared to the levels of mRNA. Incubation with low concentrations (10(-11) and 10(-10) M) of estradiol for 72 h was associated with slightly elevated levels of mRNA for ER, whereas higher concentrations gave a dose-dependent decrease. The mRNA for PR was biphasically stimulated, with a maximal effect at 10(-10)-10(-8) M, where a 10- to 13-fold stimulation was observed. The highest concentration (10(-7) M) gave a lower response. Assessment of concentration-induced variations in protein receptor levels of ER and PR reflected the effects of estradiol on their mRNAs. Low concentrations of estradiol slightly enhanced the ER level, whereas high concentrations clearly reduced ER immunoactivity. The PR level was stimulated by all concentrations used, and 10(-8) M estradiol raised the PR level more than 11-fold. Our results indicate autologous regulation of estrogen receptor gene transcripts and proteins and a clear induction of PR mRNA and receptor proteins by estradiol.

Regulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in MCF-7 cells: comparison with regulatory mechanisms of pS2 expression.

Regulation of two genes involved in tumor invasion, the matrix metalloproteinase (MMP)-1 and the tissue inhibitor of MMP (TIMP)-1, by activators of protein kinase C (PKC) or protein kinase A (PKA) was studied in MCF-7 mammary adenocarcinoma cells. The basal mRNA expression was undetectable for MMP-1 and low for TIMP-1. Treatment of MCF-7 cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) was associated with a high expression of MMP-1 mRNA, as well as an induction of the level of TIMP-1 mRNA (5- to 10-fold). In the presence of actinomycin D (AMD, 4.0 microM), an inhibitor of transcription, these stimulatory effects of TPA were abolished. Similar responses were observed when protein synthesis was inhibited by cycloheximide (CHX, 50 microM). In the presence of the cyclic AMP (cAMP) analogue N6-benzoyl (N6-Bzl)-cAMP (500 microM), the MMP-1 mRNA was unaffected and still below the level of detection, whereas a non-significant increase (< 2-fold) in TIMP-1 mRNA was observed. The level of pS2 mRNA, of which the induction by TPA in MCF-7 cells is a primary transcriptional event, was up-regulated (10- to 15-fold) by TPA (100 nM), whereas a much weaker increase (2- to 3-fold) was observed by treatment with N6-Bzl-cAMP (500 microM). Again, these stimulatory effects were counteracted by AMD (4.0 microM) and CHX (50 microM). These data suggest that activation of PKC but not of PKA may induce transcription of MMP-1 and TIMP-1, possibly by the synthesis of transcription factor(s), in transformed cells of epithelial origin.

Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells.

Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (approximately 10-fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, amongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were approximately 10-fold and approximately 15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immunoreactive PAI-1 was considerably elevated (> 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only approximately 2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (approximately 10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer.

Solid-phase differential display and bacterial expression systems in selection and functional analysis of cDNAs.

Differential gene expression can be expected during activation and differentiation of cells as well as during pathological conditions, such as cancer. A number of strategies have been described to identify and understand isolated differentially expressed genes. The differential display methodology has rapidly become a widely used technique to identify differentially expressed mRNAs. In this chapter we described a variant of the differential display method based on solid-phase technology. The solid-phase procedure offers an attractive alternative to solution-based differential display because minute amounts of sample can be analyzed in considerably less time than previously. The employed solid support, monodisperse super paramagnetic beads, which circumvents precipitation and centrifugations steps, has also allowed for optimization of the critical enzymatic and preparative steps in the differential display methodology. We also described how bacterial expression can be used as a means to elucidate gene function. An efficient dual-expression system was presented, together with a basic concept describing how parallel expression of selected portions of cDNAs can be used for production of cDNA-encoded proteins as parts of affinity-tagged fusion proteins. The fusion proteins are suitable both for the generation of antibodies reactive to the target cDNA-encoded protein and for the subsequent affinity enrichment of such antibodies. Affinity-enriched antibodies have proved to be valuable tools in various assays, including immunoblotting and immunocytochemical staining, and can thus be used to localize the target cDNA-encoded protein to certain cells in a tissue section or even to a specific cell compartment or organelle within a cell. High-resolution localization of a cDNA-encoded protein would provide valuable information toward the understanding of protein function.

Regulation of glucocorticoid receptor (GR) mRNA and protein levels by phorbol ester in MCF-7 cells. Mechanism of GR mRNA induction and decay.

Treatment of MCF-7 cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-7) M) was associated with a time-dependent increase in specific binding of [3H]dexamethasone (34.8 +/- 4.6 fmol/mg protein after 9 h of TPA treatment compared with 16.0 +/- 2.3 fmol/mg protein in control cells) as well as a transient induction in the level of glucocorticoid receptor (GR) mRNA (4- to 8-fold stimulation after 2-3 h, followed by a decline towards the control value after 6 h). In the presence of the transcription inhibitor actinomycin D (AMD) (5.0 micrograms/ml) the TPA-dependent induction of GR mRNA was completely abolished, and GR mRNA showed a gradual decline with a half-life of 2-3 h. In contrast, treatment with TPA and the protein synthesis inhibitor cycloheximide (50 microM) resulted in a superinduction of GR mRNA (> 50-fold after 6 h). Inhibition of a half-life of 2-3 h, which is identical to that observed in non-treated cells. We conclude that the increase in GR mRNA in the presence of TPA is dependent on ongoing transcription, whereas the rate by which GR transcripts are degraded, is not altered by TPA.