RESUMO
Mutations of the human Kirsten rat sarcoma viral oncogene homologue (KRAS) and the highly homologous human neuroblastoma RAS viral oncogene homologue (NRAS) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRAS and NRAS mutations, supporting their common origin. While KRAS mutations are common in colorectal cancers, NRAS mutations are relatively rare and the coexistence of multiple RAS mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of RAS. Recent experimental evidence has suggested that KRAS and NRAS may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of RAS family members and mutant forms of RAS in the colon form important considerations for the development of RAS-targeting therapeutics.
Assuntos
Adenocarcinoma/genética , Adenoma Viloso/genética , Biomarcadores Tumorais/genética , Neoplasias do Ceco/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/química , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adenoma Viloso/química , Adenoma Viloso/patologia , Adenoma Viloso/cirurgia , Biomarcadores Tumorais/análise , Biópsia , Neoplasias do Ceco/química , Neoplasias do Ceco/patologia , Neoplasias do Ceco/cirurgia , Colectomia , Análise Mutacional de DNA , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Gradação de Tumores , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Di-Hidrotestosterona/metabolismo , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Forma Celular , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Feminino , Humanos , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Androgênicos/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Testicular androgens induce the proliferation and differentiation of prostatic epithelial cells by regulating the expression of androgen target genes. The use of subtractive hybridization to isolate genes that are differentially expressed during the early phase of androgen-induced prostatic regrowth in castrated mice resulted in identification of the murine caltrin gene. Caltrin messenger RNA (mRNA) was highly expressed in the prostates of intact mice. Five weeks following castration of mice, steady state caltrin mRNA levels were reduced by 70%. Within 12 hours of administration of pharmacological doses of testosterone enanthate, steady state caltrin mRNA levels were elevated and increased to 90% of levels found in intact mice by 24 hours. Reverse transcriptase-polymerase chain reaction analysis of prostate tissue localized caltrin mRNA transcripts to the dorsal but not the ventral or lateral prostate. Within the dorsal prostate, in situ hybridization always localized caltrin mRNAs to the prostatic epithelial cells. Testosterone-induced increases in caltrin mRNA levels were detected prior to S-phase progression and initiation of proliferation in this cell population. Caltrin has been demonstrated previously to function as a calcium transport inhibitor at the plasma membrane. Findings of this study indicate that caltrin is highly expressed and androgen-regulated in the murine prostate, where it is associated with androgen-induced proliferation and differentiation of epithelial cells.
Assuntos
Androgênios/fisiologia , Camundongos/metabolismo , Próstata/metabolismo , Proteínas/genética , RNA Mensageiro/metabolismo , Animais , Masculino , Hibridização de Ácido Nucleico/métodos , Proteínas de Plasma Seminal , Distribuição TecidualRESUMO
The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.
Assuntos
Flutamida/análogos & derivados , Mutação , Receptores Androgênicos/química , Receptores Androgênicos/genética , Ativação Transcricional , Adenocarcinoma/genética , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Animais , Sítios de Ligação , Células COS , Linhagem Celular , DNA/metabolismo , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Flutamida/farmacologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese , Progesterona/farmacologia , Neoplasias da Próstata/genética , Estrutura Secundária de Proteína , Receptores Androgênicos/fisiologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
Prostate tumors were induced in Lobund-Wistar rats by treatment with N-methyl-N-nitrosourea (MNU) and testosterone propionate (TP). Androgen receptor (AR) expression was confirmed in 16 (100%) of the primary prostate cancers, with strong uniform staining in well-differentiated tumors and more variable AR immunoreactivity in poorly differentiated tumors. Epithelial cell lines were established from nine of the tumors. At early passages, four of the tumor cell lines tested were strongly immunoreactive for AR; however, only two of the cell lines, E2(A) and F2, have remained AR-positive. These cell lines specifically bind 5H-DHT at 40 and 19 fmol/mg protein, respectively, and express a 110 kDa AR immunoreactive protein. Proliferation in in vitro culture of both E2(A) and F2 cells was increased in the presence of 5alpha-dihydrotestosterone (DHT). The antiandrogen, hydroxyflutamide was able to prevent the DHT-induced growth of E2(A) but not F2 cells. Furthermore, hydroxyflutamide alone increased proliferation of F2 cells, suggesting that the androgen signalling pathway in this cell line may be abnormal. Tumorigenicity of the AR-expressing and nonexpressing cell lines was confirmed by xenograft formation following subcutaneous inoculation into intact male nude mice. In summary, carcinogen-induced prostate tumors of Lobund-Wistar rats express AR and two of nine cell lines derived from the tumors express AR. Further evaluation of AR structure in primary prostate tumors forming spontaneously or following MNU and TP induction will determine whether, as in human prostate cancers, disease progression in Lobund-Wistar rats is associated with mutations in the AR gene.
Assuntos
Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/farmacologia , Androgênios/metabolismo , Animais , Calicreínas/metabolismo , Masculino , Metilnitrosoureia/farmacologia , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/patologia , Ratos , Ratos Wistar , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Medroxyprogesterone acetate (MPA), which is frequently used as second line hormonal therapy for the treatment of metastatic breast cancer, binds with high affinity to the progesterone receptor (PR). However, the androgenic side-effects of MPA suggest that it may also activate androgen receptor (AR) regulated pathways. Treatment of the human breast cancer cell lines MDA-MB-453, ZR-75-1 and T47-D with high dose (100 nM) MPA resulted in 26-30% inhibition of cell growth, which was partially reversed by co-treatment with a 10-fold excess of the synthetic antiandrogen, anandron. Scatchard analysis demonstrated specific, high affinity (non-PR) binding of [3H]MPA to cytosols prepared from the PR-/AR+ MDA-MB-453 and PR+/AR+ ZR-75-1, but not the PR-/AR- BT-20 breast cancer cell lines. Competition of [3H]MPA binding to MDA-MB-453 cytosols by equimolar concentrations of androgens (5alpha-dihydrotestosterone (DHT), R1881) and the antiandrogen, anandron was consistent with binding of MPA to the AR. In ZR-75-1 cell cytosol fractions, DHT, R1881 and anandron only partially competed out [3H]MPA binding, suggesting that androgens displace [3H]MPA binding to AR but not to PR. Induction by MPA of AR transactivation was demonstrated in MDA-MB-453 and ZR-75-1 cells, and in the CV-1 cell line transfected with a full-length AR. In these cell lines the increased activity of the androgen responsive reporter gene (MMTV-CAT) by 1 nM MPA was fully (MDA-MB-453, CV-1) or partially (ZR-75-1) inhibited by co-culture with 1 microM anandron. These findings indicate that MPA is an AR agonist and suggest that the in vivo effects of MPA in breast cancer patients may in part be mediated by the AR.
Assuntos
Androgênios , Neoplasias da Mama/metabolismo , Imidazolidinas , Acetato de Medroxiprogesterona/metabolismo , Antagonistas de Androgênios/farmacologia , Antineoplásicos/farmacologia , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Citosol/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Acetato de Medroxiprogesterona/farmacologia , Progestinas/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Trítio , Células Tumorais CultivadasRESUMO
The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)
Assuntos
Estro/metabolismo , Glândulas Mamárias Animais/metabolismo , Receptores de Estrogênio/biossíntese , Receptores de Progesterona/biossíntese , Maturidade Sexual , Animais , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Técnicas Imunoenzimáticas , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Maintenance of the size and differentiated function of the adult prostate is dependent on testicular androgens. In this study, simultaneous androgen receptor (AR) immunohistochemistry and [(3)H]thymidine labelling was used to characterise the proliferating epithelial cells of the murine ventral prostate. Proliferation in the adult prostate was more prevalent in the basal cell population with 1.8&percent; AR-negative cells labelled with [(3)H]thymidine as compared with 0.7% AR-expressing luminal cells. Three weeks following castration of mice, the atrophied prostate contained rudimentary glands composed of both luminal and basal cells with the proportion of AR-expressing basal cells reduced from 50 to 25%. Administration of testosterone enanthate to castrated mice induced a recapitulation of the prostate gland that was preceded by up-regulation of AR expression in basal cells to normal adult levels (50% AR-positive cells) by 12 h following testosterone injection. Proliferation of AR-positive luminal cells peaked at 48 h (22.8%) while proliferation of AR-negative basal cells peaked at 96 h (6.1%) following testosterone administration. These results suggest that distinct populations of luminal and basal cells are resistant to castration-induced involution of the prostate but remain responsive to direct or indirect testosterone effects and recapitulate the gland following administration of testosterone.
Assuntos
Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Western Blotting , Contagem de Células , Divisão Celular , Células Cultivadas , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Orquiectomia , Próstata/química , Próstata/efeitos dos fármacos , Receptores Androgênicos/análise , Receptores Androgênicos/genética , Testosterona/sangue , Testosterona/farmacologia , Fatores de TempoRESUMO
PURPOSE: To determine the cellular distribution and levels of immunohistochemical staining for apolipoprotein D (Apo-D), prostate specific antigen (PSA) and androgen receptor (AR) in early stage prostate cancers. MATERIALS AND METHODS: Cellular distribution of Apo-D, PSA and AR in 30 stage A/B prostate cancers and in non-malignant glandular tissue contained in the same sections was detected immunohistochemically, and staining was evaluated by computerized video image analysis. RESULTS: Staining for Apo-D (percentage positive cellular area) was significantly increased in tumor cells of early stage prostate cancers compared with non-malignant glandular tissue. PSA and AR were present at high levels in both early stage prostate tumors and non-malignant prostate. CONCLUSIONS: Malignant transformation in the prostate is associated with increased cellular levels of Apo-D.
Assuntos
Apolipoproteínas/análise , Antígeno Prostático Específico/análise , Neoplasias da Próstata/química , Receptores Androgênicos/análise , Apolipoproteínas D , Humanos , Imuno-Histoquímica , Masculino , Estadiamento de Neoplasias , Neoplasias da Próstata/patologiaRESUMO
The mouse mammary gland undergoes rapid proliferation during puberty, then cyclical proliferation and involution during adulthood within a 5-day estrous cycle. Although proliferation of mammary epithelial cells is directed by elevated serum levels of estrogen acting via the estrogen receptor (ER), the ER status of the proliferating cells remains unknown. We examined the ER expression of proliferating epithelial cell types during pubertal development and normal adult growth using simultaneous immunohistochemistry for ER and tritiated thymidine (3H-Tdr) autoradiography. These studies demonstrate that during pubertal growth (4-6 weeks) ER-negative cells comprise more than 50% of the epithelial cell populations in the terminal end buds (TEBs) and ducts. Furthermore, the majority of proliferating cells in both TEBs and ducts are ER-negative. These findings indicate that proliferation of cells within both the TEBs and the mammary ducts contribute to pubertal growth of the mammary gland and that the greater proportion of dividing cells are ER-negative. Similar patterns of cell growth were observed in the normal estrous cycle when the majority of dividing cells were ER-negative during both pro-estrous and estrous. Intensive labelling of cells with 3H-Tdr was used to identify long-lived mammary epithelial cells which retained 3H-Tdr 2 weeks following labelling (i.e., following 3 estrous cycles). Of the small number of mammary epithelial cells retaining 3H-Tdr label, most were ER-positive luminal cells and only a few were ER-negative basal cells. This study indicates that pubertal growth of the mammary gland comprises division of ER-negative cap cells and of both ER-negative and ER-positive cells in the body of the TEBs and elongating mammary ducts. Similarly, estrogen-driven proliferation of ER-negative and ER-positive luminal cells and ER-negative basal cells maintains the differentiated mammary gland in the adult mouse.
Assuntos
Células Epiteliais/química , Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptores de Estrogênio/análise , Maturidade Sexual/fisiologia , Animais , Autorradiografia , Divisão Celular/fisiologia , Feminino , Imuno-Histoquímica , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ovário/fisiologiaRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and is expressed at elevated levels in several tumor types. In this study immunohistochemical localization and distribution of isoforms of VEGF were examined in malignant and non-malignant human prostatic tissues. MATERIALS AND METHODS: Immunohistochemical localization of VEGF was performed on thirty well, moderately and poorly differentiated stage D2 prostate cancer specimens and twenty benign prostatic hyperplasia (BPH) specimens. VEGF mRNA was determined by polymerase chain reaction and VEGF protein isoforms were detected by Western blotting of prostate cancer and BPH specimens. RESULTS: Cytoplasmic immunoreactivity for VEGF was detected in tumor cells and peritumoral stromal cells of prostate cancer specimens and in non-malignant glandular epithelial cells and interglandular stromal cells in BPH specimens. Staining was focal with areas of strongly to weakly stained cells adjacent to negatively staining areas. mRNA's for VEGF121, VEGF165 and VEGF189 were present in all benign and malignant prostate specimens. VEGF protein isoforms of molecular sizes corresponding to VEGF165 and VEGF189 were detected in cytosolic extracts of prostate cancers and BPH specimens by Western blotting. In addition, two novel higher molecular weight immunoreactive bands were detected in the prostate specimens. CONCLUSIONS: Widespread distribution of VEGF in prostate cancers and BPH specimens suggest that the VEGF165, VEGF189 isoforms and novel 90 and 110 kD forms detected may contribute to the establishment or progression of these conditions.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Western Blotting , Humanos , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Little is known regarding the activity and function of the androgen receptor (AR) in human breast cancer. In the present study AR was evaluated in untreated primary breast cancers using antisera to the amino- and carboxy-termini of the receptor and quantitated using colour video image analysis. A strong correlation between tissue concentration and percentage AR-positive cells was observed for each antiserum. However, comparison of percentage positive cells using the amino- and carboxy-terminal AR antisera in individual breast cancer specimens revealed a subset of tumours with discordantly increased staining for the carboxy terminus. These findings suggest the presence of amino-terminal-truncated AR in a proportion of breast cancer cells or presence of AR mutations or associated protein alterations that affect binding of the amino-terminal AR antiserum. Immunohistochemical expression of the androgen-regulated glycoprotein, apolipoprotein D (apo-D), was also evaluated in the breast cancer specimens. Focal positivity of apo-D staining, which did not always co-localise with AR-positive cells, was observed within breast tumours. Furthermore, no correlation was evident between percentage positive cells stained for AR and apo-D in breast cancer specimens. These findings indicate that, although apo-D expression is androgen regulated in human breast cancer cell lines in vitro, its expression in primary breast cancers may be regulated by other factors. The expression of AR in primary breast cancers also suggests that the receptor may be involved in tumour responsiveness or in abnormal responses to endocrine therapies.
Assuntos
Apolipoproteínas/análise , Neoplasias da Mama/química , Neoplasias da Mama/ultraestrutura , Proteínas de Neoplasias/análise , Receptores Androgênicos/análise , Adulto , Idoso , Apolipoproteínas D , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica , Microscopia de Vídeo/métodos , Pessoa de Meia-IdadeRESUMO
Progression to androgen-independent growth of human prostate cancers may be mediated by alterations in the structure and/or expression of the androgen receptor (AR) gene. To date, mutations in the AR gene have largely been identified in hormone refractory tumors. In this study, single-strand conformational polymorphism analysis and DNA sequencing of the entire AR gene coding region was performed on 25 primary prostate tumors sampled prior to initiation of hormonal (i.e. , androgen ablation) therapy. Base changes leading to amino acid substitutions in the AR were identified in 11 (44%) tumors. The presence of AR amino acid substitutions was associated with decreased immunohistochemical staining for AR in tumor cells and the rapid failure of subsequent hormonal therapies. Single-strand conformational polymorphism analysis of exons 2, 3, and 8 of the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) gene in the same samples revealed no bandshifts, suggesting that the high frequency of AR gene mutations detected was not a consequence of generalized genetic instability. These data indicate that AR gene mutations occur commonly in advanced prostate cancers prior to endocrine treatment of disease and may contribute to altered androgen responsiveness of the tumors.
Assuntos
Androgênios/fisiologia , Mutação , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética , DNA/química , Humanos , Hipoxantina Fosforribosiltransferase/genética , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/análise , Sequências Repetitivas de Ácido Nucleico , Células Tumorais CultivadasRESUMO
Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I, IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (> or = 10(-6) M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10(-7) M RA, and that 10(-9) - 10(-7) M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10(-9) - 10(-7) M RA. Treatment with the IGF-I receptor blocking antibody, alpha IR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10(-9) - 10(-7) M RA enhancing cell proliferation and > or = 10(-6) M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies.
Assuntos
Neoplasias da Mama/patologia , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Tretinoína/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ensaio Radioligante , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Tretinoína/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
PURPOSE: To investigate Apolipoprotein-D (Apo-D) and prostate specific antigen (PSA) immunohistochemical staining of nonmalignant and malignant human prostate tissues. MATERIALS AND METHODS: Apolipoprotein-D and PSA immunoreactivity were evaluated by video image analysis in nonmalignant prostates and in 30 stage D2 prostate cancers. RESULTS: Apolipoprotein-D was detected in all 30 tumors, and the level of staining was elevated in comparison to age-matched nonmalignant prostates (p < 0.05). In contrast, the level of PSA staining in tumors was less than that detected in nonmalignant prostates. CONCLUSIONS: Apolipoprotein-D is expressed in normal human prostate. Elevated Apo-D staining is associated with advanced prostate cancer.
Assuntos
Apolipoproteínas/biossíntese , Antígeno Prostático Específico/biossíntese , Doenças Prostáticas/metabolismo , Neoplasias da Próstata/metabolismo , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas D , Criança , Pré-Escolar , Humanos , Lactente , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: To determine the predictive value of androgen receptor (AR) levels in primary tumors of women who undergo medroxyprogesterone acetate (MPA) therapy for advanced breast cancer after relapse on tamoxifen adjuvant therapy. METHODS: Between 1984 and 1987 at Flinders Medical Centre, South Australia, 136 postmenopausal women received adjuvant tamoxifen therapy for lymph node-positive breast cancer. Estrogen receptor (ER), progesterone receptor (PgR), and AR levels, tumor size, and degree of axillary node involvement were determined at the time of diagnosis. The median follow-up period was 81 months; 89 women developed metastatic disease, 83 of whom subsequently received MPA (500 mg/d). The objective response rate ([RR] ie, complete response [CR] and partial response [PR]) and progression-free interval (PFI) were assessed in response to MPA therapy. Associations between RR, PFI, and primary tumor characteristics including ER, PgR, and AR levels were examined using the Mann-Whitney U test, Kaplan-Meier product-limit estimator, and Cox proportional hazards regression, as appropriate. RESULTS: Thirty-two of 83 patients (38.6%) responded to MPA. RR was significantly associated with the presence of AR (P < .001), but not with other primary tumor characteristics or duration of tamoxifen therapy. After initiation of MPA treatment, PFI increased with increasing concentration of AR in the primary tumor. CONCLUSION: Response to MPA after adjuvant tamoxifen treatment for lymph node-positive breast cancer was positively associated with AR level in the primary tumor. This finding suggests that MPA action in breast cancer may be mediated in part by the AR.
Assuntos
Neoplasias da Mama/química , Acetato de Medroxiprogesterona/uso terapêutico , Receptores Androgênicos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Tamoxifeno/uso terapêutico , Falha de TratamentoRESUMO
Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.
Assuntos
Androgênios/farmacologia , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Imidazolidinas , Antagonistas de Receptores de Andrógenos , Di-Hidrotestosterona/farmacologia , Flutamida/análogos & derivados , Flutamida/farmacologia , Humanos , Imidazóis/farmacologia , Nandrolona/análogos & derivados , Nandrolona/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Células Tumorais CultivadasRESUMO
Progression to hormone-refractory disease is a common outcome of human prostate cancer. In this study, we have investigated the basis of androgen insensitivity in the human prostate cancer cell line, PC-3, which was derived from a bone metastasis of a hormone-refractory prostate cancer. PC-3 cells with virtually undetectable (PC-3AR-) or low (PC-3AR+) levels of androgen receptor (AR) RNA expression were examined. RNase protection assays demonstrated that the level of AR RNA in PC-3AR+ cells was similar to that in a normal androgen-responsive genital skin fibroblast cell strain. Quantitative immunocytochemical staining of AR in PC-3AR+ cells using antibodies directed against the amino and carboxyl termini of the receptor revealed staining in 30% of cells with either antibody. Furthermore, the level of AR staining in PC-3AR+ cells was higher than in the androgen-responsive breast cancer cell lines ZR-75-1, T47-D, and MCF-7. Despite the expression of AR RNA and protein, PC-3AR+ cell proliferation was unaffected by the presence of 0.1-10 nM mibolerone. Scatchard analysis demonstrated a complete absence of specific [3H]dihydrotestosterone ([3H]DHT) binding to PC-3AR+ cytosolic extracts, which could not be explained by structural alterations in the AR gene. The sizes of individual AR exons amplified from genomic DNA derived from PC-3AR+ cells were identical to those amplified from normal human cells. Furthermore, sequence analysis did not reveal a mutation in the DNA- or hormone-binding domains of the AR gene in PC-3AR+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Androgênios/farmacologia , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/tratamento farmacológico , RNA Neoplásico/biossíntese , Receptores Androgênicos/genética , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/patologia , Ensaio Radioligante , Células Tumorais CultivadasRESUMO
All-trans retinoic acid (tRA) inhibits growth of estrogen receptor-positive (ER+) breast cancer cells in vitro, and a variety of retinoids inhibit development of breast cancer in animal models. 9-cis retinoic acid (9-cis RA) is a naturally occurring high affinity ligand for the retinoid X receptors, as well as the retinoic acid receptors (RARs). Whether 9-cis RA has a different spectrum of biological activity from tRA, which only binds RARs with high affinity, is largely unknown. We studied the effects of 9-cis RA on growth and gene expression in ER+ and ER- human breast cancer cells. 9-cis RA inhibited the growth in monolayer culture of several ER+, but not ER-, cell lines in a dose-dependent manner. Growth inhibition and morphological changes by 9-cis RA were similar to those of tRA, suggesting that the ability to bind both RAR and retinoid X receptors did not significantly augment growth inhibition or confer sensitivity to tRA-resistant lines. MCF-7 cells exposed to 9-cis RA showed a dose-dependent accumulation in G1. Northern analyses showed that RAR-alpha and RAR-beta were not significantly regulated, while RAR-gamma was up-regulated and retinoid X receptor alpha was down-regulated by 9-cis RA. Since interactions between tRA and ER-dependent transcription have recently been reported, we investigated whether these retinoids regulate expression of ER itself or estrogen-responsive genes. Both 9-cis RA and tRA induce down-regulation of ER mRNA and protein in MCF-7 cells. 9-cis RA down-regulates expression of the estrogen-responsive genes PR and pS2 in MCF-7 cells as reported previously for tRA. In several ER-positive subclones, we found that the degree of ER expression and regulation, but not always estrogen-sensitivity, correlates with the growth-inhibitory effects of 9-cis RA. Further, in an ER-, retinoid-unresponsive breast cancer cell line, induced ER expression confers responsiveness to retinoid growth inhibition. These data, combined with reports of additive growth inhibition of tRA and tamoxifen in vitro, suggest that 9-cis RA might augment the ability of tamoxifen to inhibit growth of ER+ breast cancer cells in vivo.