RESUMO
BACKGROUND: We assessed sperm DNA fragmentation index (DFI) in cancer patients before and after treatment to evaluate if sperm DNA integrity is compromised by cancer itself or its treatment. METHODS: In a prospective study, DFI was assessed in 127 patients diagnosed with testicular germ cell tumours (TGCT), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL) and various malignancies. The severity of cancer and tumour markers at diagnosis was recorded. Follow-up DFI after treatment was available in 52 patients who were mostly less severely affected. RESULTS: In patients diagnosed with TGCT, HL and various malignancies, pretreatment DFI levels were not significantly different from that of proven fertile controls, but in patients with NHL an increased DFI was found. An overall significant decrease in post-treatment DFI (13.2% range 5.0-70.5) compared with pretreatment values (17.1% range 5.1-66.6) was found (P = 0.040). In TGCT patients, post-treatment DFI was significantly higher in patients who were treated with radiotherapy (16.9% range 11.5-39.9) compared with that in patients treated with chemotherapy (CT) alone (10.9% range 5.5-39.9) (P = 0.037). In HL patients, the type of treatment or number of CT cycles was not associated with DFI. Overall, post-treatment DFI in cancer patients was not significantly different from that of proven fertile controls. CONCLUSIONS: In this study, the presence of cancer does not seem to negatively affect the sperm DNA integrity in TGCT and HL patients; only NHL patients showed increased DFI at the time of diagnosis compared with healthy controls. Our results confirm previous reports that DFI decreases significantly following various anti-cancer treatments. In contrast, radiotherapy in TGCT patients is associated with an increase in DFI compared with CT treatment alone.
Assuntos
Antineoplásicos/efeitos adversos , Fragmentação do DNA , Espermatozoides/efeitos dos fármacos , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/radioterapia , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/radioterapia , Masculino , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/radioterapia , Análise do Sêmen , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/radioterapiaRESUMO
Immunosuppression was investigated to enhance the take rate of various human tumor types in nude BALB/c or B10.LP/Cpb mice. Cyclophosphamide (CY), known as an immunosuppressive agent, was injected ip at a dose of 100 mg/kg 24 hours prior to subcutaneous implantation of tumor fragments obtained from established xenograft lines or from patients. In 3 out of 8 ovarian adenocarcinoma tumor lines, with a take of 50% or less in untreated control animals, tumor take was significantly increased by CY treatment to values between 75 and 100%. No effect of CY treatment on tumor take was seen with squamous cell carcinomas of the head and neck region and with lung and prostatic carcinomas. The growth rate of these xenografts was not affected by CY pretreatment. These results indicate that immunologic mechanisms indeed can play a role in the inhibition of tumor growth in nude mice. CY pretreatment may enhance the success rate of transplantation, but this effect appears to be limited to certain tumors.
Assuntos
Adenocarcinoma/imunologia , Ciclofosfamida/imunologia , Neoplasias Ovarianas/imunologia , Adenocarcinoma/patologia , Animais , Divisão Celular , Linhagem Celular , Ciclofosfamida/administração & dosagem , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Neoplasias Ovarianas/patologia , Imunologia de TransplantesRESUMO
In an attempt to define a biochemical marker of ornithine decarboxylase inhibition in humans, alpha-difluoromethylornithine hydrochloride (DFMO), an irreversible ornithine decarboxylase inhibitor, was infused i.v. in seven cancer patients over 10-day courses at doses of 10-90 g/day and 24-h urinary excretion of polyamines and decarboxylated-S-adenosylmethionine was determined before, during, and after treatment. DFMO produces marked increases in urinary decarboxylated-S-adenosylmethionine excretion, up to 84 times pretreatment values. This response appears to be time dependent, requiring several days to reach a maximum and lasting at least 4-5 days after stopping DFMO. In contrast, urinary excretion of the polyamines putrescine, cadaverine, spermidine, N1-monoacetylspermidine, N8-monoacetylspermidine, and spermine, were not consistently altered by DFMO. We conclude that urinary excretion of decarboxylated-S-adenosylmethionine represents a valid biochemical indicator of ornithine decarboxylase inhibition in humans, whereas urinary polyamines are of no value.
Assuntos
Eflornitina/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores da Ornitina Descarboxilase , S-Adenosilmetionina/análogos & derivados , Idoso , Creatinina/urina , Eflornitina/farmacologia , Eflornitina/urina , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Neoplasias/enzimologia , Neoplasias/urina , Poliaminas/urina , S-Adenosilmetionina/urinaRESUMO
The results described in this paper demonstrate that proliferation arrest by low concentrations of tetracyclines, which has previously been shown in experiments with animal tumor systems, can also be achieved in tumor systems of human origin. Tetracyclines specifically inhibit mitochondrial protein synthesis. Prolonged and continuous impairment of protein synthesis inside the mitochondria leads to reduction of the cellular concentration of the polypeptide products which are coded and synthesized within mitochondria. These products are part of the oxidative phosphorylative system of the cell. Long-term tetracycline treatment leads to a decrease of oxidative ATP-generating capacity as monitored by cytochrome c oxidase activity. This may cause severe energetic or metabolic disturbances which explain the proliferation arrest observed. Proliferation arrest, provided that mitochondrial protein synthesis is blocked effectively, is found in vitro as well as in vivo. It is shown that the effect of doxycycline is not limited to cytostasis; prolonged doxycycline treatment is clearly cytotoxic for the tumor cells.
Assuntos
Antineoplásicos , Doxiciclina/farmacologia , Neoplasias Renais/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Doxiciclina/uso terapêutico , Esquema de Medicação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Biossíntese de Proteínas , Ratos , Análise EspectralRESUMO
Light-induced absorbance changes were measured at low temperatures in reaction center preparations from Rhodopseudomonas sphaeroides. Absorbance difference spectra measured at 100 degrees K show that ubiquinone is photoreduced at this temperature, both by continuous light and by a short actinic flash. The reduction occurred with relatively high efficiency. These results give support to the idea that ubiquinone is involved in the primary photochemical reaction in Rhodopseudomonas sphaeroides. Reduction of ubiquinone was accompanied by a shift of the infrared absorption band of bacteriopheophytin. The rate of decay of the primary photoproducts (P+870 and ubisemiquinone) appeared to be approximately independent of temperature below 180 degrees K and above 270 degrees K; in the region between 180 and 270 degrees K it increased with decreasing temperature. The rate of decay was not affected by 0-phenanthroline. Secondary reactions were inhibited by lowering the temperature. The light-induced absorbance changes were inhibited by chaotropic agents, like thiocyanate and perchlorate. It was concluded that these agents lower the efficiency of the primary photoconversion. The kinetics indicated that the degree of inhibition was not the same for all reaction centers. The absorption spectrum of the photoconverted reaction centers appeared to be somewhat modified by thiocyanate.
Assuntos
Acetatos/farmacologia , Fluoracetatos/farmacologia , Guanidinas/farmacologia , Iodetos/farmacologia , Percloratos/farmacologia , Rhodobacter sphaeroides , Rhodobacter sphaeroides/metabolismo , Tiocianatos/farmacologia , Ureia/farmacologia , Escuridão , Congelamento , Luz , Fenantrolinas/farmacologia , Fotoquímica , Rhodobacter sphaeroides/efeitos dos fármacos , Espectrofotometria , Espectrofotometria UltravioletaRESUMO
(1) A flash number dependency of flash-induced absorbance changes was observed with whole cells of Rhodospirillum rubrum and chromatophores of R. rubrum and Rhodopseudomonas sphaeroides wild type and the G1C mutant. The oscillatory behavior was dependent on the redox potential; it was observed under oxidizing conditions only. Absorbance difference spectra measured after each flash in the 275--500 nm wavelength region showed that a molecule of ubiquinone, R, is reduced to the semiquinone (R-) after odd-numbered flashes and reoxidized after even-numbered flashes. The amount of R reduced was approximately one molecule per reaction center. (2) The flash number dependency of the electrochromic shift of the carotenoid spectrum was studied with chromatophores of Rps. sphaeroides wild type and the G1C mutant. At higher values of the ambient redox potential a relatively slow phase with a rise time of 30 ms was observed after even-numbered flashes, in addition to the fast phase (completed within 0.2 ms) occurring after each flash. Evidence was obtained that the slow phase represents the formation of an additional membrane potential during a dark reaction that occurs after flashes with an even number. This reaction is inhibited by antimycin A, whereas the oscillations of the R/R- absorbance changes remain unaffected. At low potentials (E = 100 mV) no oscillations of the carotenoid shift were observed: a fast phase was followed by a slow phase (antimycin-sensitive) with a half-time of 3 ms after each flash. (3) The results are discussed in terms of a model for the cyclic electron flow as described by Prince and Dutton (Prince, R.C. and Dutton, P.L. (1976) Bacterial Photosynthesis Conference, Brussels, Belgium, September 6--9, Abstr. TB4) employing the so-called Q-cycle.
Assuntos
Fotossíntese , Rhodobacter sphaeroides/metabolismo , Rhodospirillum rubrum/metabolismo , Ubiquinona/metabolismo , Cromatóforos Bacterianos/metabolismo , Escuridão , Transporte de Elétrons , Cinética , Luz , Oxirredução , Especificidade da Espécie , EspectrofotometriaRESUMO
Human prostate-specific transglutaminase (hTG(P)) is a cross-linking enzyme encoded by the TGM4 gene. The TGM4 gene promoter was characterized by deletion mapping and mutational analysis. Promoter constructs, containing the minimal promoter requirements, could efficiently drive transcription in the prostate cancer cell lines PC346C and LNCaP and the hepatic cancer cell line Hep3B. The region between positions -113 and -61 was demonstrated to be essential for core promoter activity. Further analysis revealed the functional importance of an Sp1 binding motif, 5'-ACCCCGCCCC-3', at positions -96 to -87. This sequence is a binding site of the ubiquitous transcription factors Sp1 and Sp3.
Assuntos
Regiões Promotoras Genéticas/genética , Próstata/enzimologia , Fator de Transcrição Sp1/metabolismo , Transglutaminases/genética , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Fator de Transcrição Sp3 , Fatores de Transcrição/metabolismo , Transglutaminases/química , Transglutaminases/metabolismo , Células Tumorais CultivadasRESUMO
Using a prostate-specific antigen cDNA as a hybridization probe, clones containing the kallikrein genes encoding prostate-specific antigen, human glandular kallikrein-1 and pancreas/kidney kallikrein were isolated from a human genomic library. Clones containing the prostate-specific antigen gene and the human glandular kallikrein-1 gene overlap and span a region of about 36 kb. The two genes are aligned in a head to tail orientation at a mutual distance of 12 kb. Southern blot analysis of DNA from a panel of human-hamster hybrid cells with specific probes revealed the genes to be situated on chromosome 19. Assuming that the pancreas/kidney kallikrein gene is located in the same cluster, the distance to the prostate-specific antigen gene and the human glandular kallikrein gene must be at least 15 kb.
Assuntos
Antígenos de Neoplasias/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Calicreínas/genética , Animais , Clonagem Molecular , Cricetinae , Sondas de DNA , Desoxirribonuclease BamHI , Desoxirribonuclease HindIII , Humanos , Células Híbridas , Rim/enzimologia , Masculino , Hibridização de Ácido Nucleico , Pâncreas/enzimologia , Próstata , Antígeno Prostático Específico , Calicreínas TeciduaisRESUMO
The aim of this study was to obtain insight into the role of the multidrug resistance (MDR) phenomenon in hormone-independent progressive prostate cancer. Using immunocytochemistry and Western blotting we determined the expression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi (GST-pi), Bcl-2, Bax, topoisomerase (Topo) I, II alpha and II beta in the human prostate cancer cell lines PC3, TSU-Pr1, DU145 and LNCaP derivatives LNCaP-R, LNCaP-LNO and LNCaP-FGC. Proliferative activity was assessed by immunocytochemistry. MTT assays were used to determine the sensitivity to etoposide, doxorubicin and vinblastin. Pgp was not expressed in any of the cell lines. MRP was variably expressed. GST-pi was expressed in TSU-Pr1, PC3 and DU145. The expression of Bcl-2 was restricted to TSU-Pr1, whereas Bax was found in all cell lines. Topo II alpha was expressed at the highest level in the rapidly proliferating cell lines TSU-Pr1 and DU145. Topo I and II beta were equally expressed. Resistance profiles varied among the cell lines, with TSU-Pr1 being the most sensitive and LNCaP-LNO relatively resistant. Multiple MDR proteins were expressed in prostate cancer cell lines and may well influence response to chemotherapy. Future functional studies, using chemo-selected MDR models, may further help to determine the mechanism or combination of mechanisms underlying the resistance of prostate cancer to chemotherapy.
Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Western Blotting , DNA Topoisomerases Tipo I/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Human peritoneal macrophages collected from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during inflammation-free periods were induced to express antitumor activity in vitro when cultured in the presence of bacterial lipopolysaccharide (LPS) and even more activity when they were kept in the presence of LPS + IND (indomethacin). The antitumor activity was expressed against a human tumor-cell line, RC43, either in a cell-to-cell contact set-up between the macrophages and the RC43 target cells or when the tumor cells were exposed to supernatants of the cultured macrophages. The antitumor activity of macrophages was correlated to a marked increase in production of tumor necrosis factor-alpha (TNF alpha), not correlated to an increase in nitrite production and inversely correlated to the production of PGE2. The RC43 tumor cells were susceptible to recombinant human TNF alpha, recombinant human IL-1 beta, sodium nitrite and the leukotriene LTB4. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.
Assuntos
Carcinoma de Células Renais/imunologia , Dinoprostona/biossíntese , Neoplasias Renais/imunologia , Macrófagos Peritoneais/imunologia , Nitritos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Meios de Cultura , Citotoxicidade Imunológica/imunologia , Humanos , Indometacina/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Nus , Diálise Peritoneal Ambulatorial Contínua , Células Tumorais CultivadasRESUMO
The involvement of mitochondrial damage in the antiproliferative effects of m-iodobenzylguanidine [MIBG] and methylglyoxal bis (guanylhydrazone) [methylGAG] was studied in human neuroblastoma SK-N-SH, mouse neuroblastoma N1E115 and mouse lymphosarcoma S49 cells. Proliferation of SK-N-SH cells was insensitive to MIBG (100 microM gave 15% inhibition), but sensitive to methylGAG (IC50 = 50 microM). MIBG and methylGAG were approximately equitoxic to N1E115 cells (IC50 of 92 and 87 microM, respectively). S49 cells were most sensitive to both MIBG (IC50 = 11 microM) and methylGAG (IC50 = 5 microM). In isolated sonicated mitochondria, MIBG inhibited respiration a complex I of the respiratory chain (EC50 = 0.5 mM), whereas methylGAG was much less effective (EC50 greater than 15 mM). In intact cells, MIBG at 31 microM impaired mitochondrial respiration and stimulated the glycolytic flux. In contrast, equimolar concentrations of methylGAG had no effect on oxygen consumption, ATP content, glucose consumption and lactate production. MethylGAG significantly increased putrescine levels in N1E115 and S49 cells within 12 hr via inhibition of S-adenosylmethionine decarboxylase. No such effects were seen in SK-N-SH cells for up to 48 hr. Equimolar concentrations of MIBG had no effect on the putrescine levels in the various cell lines, suggesting that MIBG did not inhibit S-adenosylmethionine decarboxylase. It is concluded that the antiproliferative mechanisms of the guanidino compounds are essentially different. MIBG inhibited mitochondrial respiration at complex I with concomitant stimulation of the glycolytic flux but was essentially without effect on polyamine levels. On the other hand, cytotoxicity of methylGAG was not associated with mitochondrial dysfunction.
Assuntos
Antineoplásicos/farmacologia , Iodobenzenos/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Mitoguazona/farmacologia , Neuroblastoma/tratamento farmacológico , 3-Iodobenzilguanidina , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Líquido Intracelular/metabolismo , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poliaminas/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The screening of an oligo(dT)-primed prostate cDNA library with a human glandular kallikrein-1 (hGK-1) genomic DNA fragment resulted in the isolation of two different hGK-1 cDNAs. A 1.2 kb cDNA (pGK-1) contains an open reading frame of 510 bp, encoding the major part of the previously predicted hGK-1 protein (Schedlich et al. (1987) DNA 6, 429-437). This cDNA contains a 3'-untranslated region of 677 nucleotides and terminates in a poly(A) stretch, preceded by the canonical AATAAA polyadenylation signal. A second cDNA (pGK-10A), with a size of 1.5 kb, contains an open reading frame of 669 nucleotides preceded by 16 nucleotides of the 5'-untranslated region. pGK-10A differs from pGK-1 by the presence of an additional 37 bp fragment, interrupting the protein coding region of hGK-1, which results from the use of an alternative splice donor site of intron IV of the hGK-1 gene. The mature protein (excluding presumed pre- and propeptides) as deduced from the pGK-10A cDNA sequence, has a size of 199 amino acids and differs at the COOH-terminus from the 237 amino acid hGK-1 protein. The alternatively spliced mRNA comprises approximately 20% of the hGK-1 transcripts, as deduced from analysis of mRNA from prostate cells by PCR amplification of specific fragments. The regulation of hGK-1 mRNA expression was studied in different human prostate tumors and cell lines by Northern blotting, using a hGK-1-specific oligonucleotide probe. A high level of hGK-1 expression was found in the androgen-dependent tumors PC 82 and PC EW. hGK-1 mRNA was also present in the androgen-sensitive LNCaP cell line, but undetectable in the androgen-insensitive prostate tumors PC 133, PC 135 and the PC 3 cell line. In LNCaP cells, the expression of hGK-1 mRNA was strongly induced by androgens. Regulation of expression of the closely related prostate-specific antigen (PA) gene showed a similar pattern.
Assuntos
Androgênios/fisiologia , Regulação da Expressão Gênica , Calicreínas/genética , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Northern Blotting , DNA , Humanos , Calicreínas/biossíntese , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Antígeno Prostático Específico , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
Fluorescence in situ hybridization (FISH) with centromere probes was used to investigate numerical aberrations of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate carcinoma (PC) and 11 benign prostatic hyperplasia (BPH) samples. None of the benign specimens showed any chromosomal aberration. Forty-one of 46 PC specimens showed numerical aberrations of one or more chromosomes. All investigated chromosomes showed numerical aberrations in at least 30% of the specimens, gain being more frequent than loss. Comparison of DNA flow cytometry (FCM) and FISH results showed that not only aneuploid tumors but also most diploid tumors harbored numerical chromosome aberrations. Chromosome 10 was the most frequently gained (65%), and Y the most frequently lost chromosome (14%). Nonmetastatic and metastatic tumors differed significantly (P < .05) in the number of copies for chromosomes 7, 8, and 10, but not for 1, 18, and Y. These results suggest strongly that gains of chromosomes 7, 8, and 10 are involved in PC progression.
Assuntos
Aneuploidia , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 10 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 8 , DNA de Neoplasias/genética , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Hiperplasia Prostática/genética , Neoplasias da Próstata/patologia , Cromossomo YRESUMO
Renal cell carcinoma (RCC) displays strong resistance against many chemotherapeutic drugs. Overexpression of P-glycoprotein (Pgp) appears to be part of this resistance. The involvement of another resistance mechanism, involving the decreased activity of DNA topoisomerase II (topoII), remains uncertain. By culturing the human RCC lines RC2 and RC21 in the presence of increasing concentrations of etoposide, we derived the variant sublines RC2E, RC21A and RC21E, that had acquired approximately 30-, 60- and 90-fold resistance to this drug respectively. RC2E, RC21A and RC21E were approximately 50-, 5- and 400-fold cross-resistant to doxorubicin respectively. RC2E and RC21E also showed cross-resistance (approximately 200- and 3500-fold respectively) to vinblastine. Quantitative differences in MDR1 and Pgp expression (elevated in RC2E and RC21E) and topoII alpha (reduced in RC21E and RC21A) were demonstrated using Western blotting and the reverse transcriptase/polymerase chain reaction. Decreased amounts of topoII alpha were reflected in a reduced activity of RC21A and RC21E as measured by unknotting phage P4 DNA. Qualitative changes of the topoII alpha gene, such as point mutations in the motif B/DNBS and DNA-binding regions, or differences in methylation status of the promoter gene of RC21E, were not found. These cell lines represent a model of a solid tumor in which overexpression of Pgp, a combination of increased Pgp and decreased topoII alpha, and a decrease of topoII alpha are represented.
Assuntos
Carcinoma de Células Renais/enzimologia , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/farmacologia , Isoenzimas/metabolismo , Neoplasias Renais/enzimologia , Antígenos de Neoplasias , Antineoplásicos/farmacologia , Metilação de DNA , Proteínas de Ligação a DNA , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica/efeitos dos fármacos , Humanos , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais Cultivadas , Vimblastina/farmacologiaRESUMO
The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT codon encoding a threonine residue to GCT, encoding alanine.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Detailed cytogenetic analysis was performed of a xenografted human prostatic adenocarcinoma cell line PC-82. A direct preparation method was developed that yielded metaphases of good quality. Flow cytometric data and banding analysis of metaphases showed a near-tetraploid karyotype with 18 consistent marker chromosomes. As a result of the rearrangements involved, parts of chromosomes 2, 3, 4, 7, 9, 10, 15, 18, and 21 were homozygous, while regions on 2p, 13q, and 17q were apparently completely lost. In contrast to this, some regions on #2, #5, and, especially, on #1 were present in three or even four times the normal copy number. Comparison of affected chromosomes in PC-82 with all data available on prostatic carcinoma showed chromosomes 1, 2, 3, 6, 7, 10, and 15 to be involved in rearrangements in over 50% of all prostatic carcinomas. When only data from primary prostatic adenocarcinomas (including PC-82) were taken into account it appeared that chromosomes 1, 7, and 10 were involved in all five primary tumors studied.
Assuntos
Adenocarcinoma/genética , Aberrações Cromossômicas , Neoplasias da Próstata/genética , Adenocarcinoma/patologia , Animais , Linhagem Celular , Bandeamento Cromossômico , Citometria de Fluxo , Humanos , Cariotipagem , Masculino , Metáfase , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias da Próstata/patologia , Transplante HeterólogoRESUMO
Karyotypic analysis was performed on 102 prostate cancer specimens which were obtained through radical prostatectomy, transurethral resection, or regional lymph node dissection. Short term tissue culture was applied in all cases. Of the media and growth factors evaluated, F12/DMEM, supplemented with 2% fetal calf serum, insulin, epidermal growth factor, hydrocortisone, and cholera toxin produced the largest increase of in vitro proliferation. Such in vitro cultured cells were all phenotypically acinar epithelial cells, the supposed targets for neoplastic transformation. Stromal cell growth appeared to be completely suppressed. Of the three culture techniques investigated, the method developed in Lund, Sweden, was the most successful: 11/15 cultures yielded metaphases and, in three of these, clonal aberrations were identified. All 39 karyotypes obtained essentially had a 46,XY karyotype with clonal aberrations (eight cases) and/or nonclonal aberrations (30 cases). Clonal structural aberrations involved 2p, 3q, 11p, 17p, and 21q. The clonal numerical aberrations found were: + 8, + dmin, and -Y. The most frequently observed nonclonal aberrations were 8p deletions (five cases) and loss of 6, 7, 8, 15, 17, 18, 21, and/or Y (> or = five cases). In summary, clonal aberrations were observed in 20% of the evaluable PC cell cultures, and nonclonal aberrations in 77%. So, although diploid cells without clonal abnormalities still had a growth advantage, under optimal conditions PC cells were able to proliferate in primary in vitro culture.
Assuntos
Carcinoma/genética , Técnicas de Cultura/métodos , Cariotipagem/métodos , Neoplasias da Próstata/genética , Carcinoma/química , Carcinoma/patologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Estudos de Avaliação como Assunto , Humanos , Imuno-Histoquímica , Queratinas/análise , Masculino , Antígeno Prostático Específico/análise , Neoplasias da Próstata/química , Neoplasias da Próstata/patologiaRESUMO
Using chromosome banding and fluorescence in situ hybridization (FISH) with painting probes, sequential cytogenetic analysis was performed of two novel prostate cell lines, PZ-HPV-7 and CA-HPV-10, established by human papillomavirus (HPV) 18 DNA transformation. PZ-HPV-7 originates from a normal diploid prostate epithelial cell strain. PZ-HPV-7 progressed from an initial diploid to a hypertetraploid chromosome number with a relative gain of chromosomes 5 and 20 (7 to 8 copies each). Structural changes were limited; 3p- (2 copies), 3q- (1 copy), and possibly a der(16p;12q). CA-HPV-10 originates from an epithelial cell strain derived from a high-grade human prostate cancer specimen, which showed several karyotypic abnormalities including an extra Y chromosome and double minutes (dmin). In early passage the karyotype of CA-HPV-10 appeared unstable with a decreasing number of cells exhibiting dmin. In late passage the dmin were replaced by a large homogeneously staining region (hsr) on 9p+ marker. The hsr was shown by FISH to be of chromosome 1 origin. The modal number was mainly hypertriploid (72, range 69 to 75). Loss of Y was remarkable (0 to 1 copy). Consistent markers included two copies each of del(1)(q12q31) and der(9)t(1;9)(?;p22), and one der(11)t(4;11) (?;q21). HPV type 18 genomic integration sites were identified on 1p for PZ-HPV-7 and on the 9p+ marker for CA-HPV-10. In conclusion, both PZ-HPV-7 and CA-HPV-10 showed clonal cytogenetic changes. These two cell lines constitute a novel in vitro model to study the mechanisms involved in human prostate carcino-genesis.
Assuntos
Linhagem Celular Transformada , Aberrações Cromossômicas , DNA Viral , Papillomaviridae/genética , Neoplasias da Próstata/genética , Bandeamento Cromossômico , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Ploidias , Transfecção , Células Tumorais CultivadasRESUMO
OBJECTIVES: To investigate expression of the prostatic markers prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM), and the androgen receptor (AR) after human papillomavirus (HPV) type 18 deoxyribonucleic acid (DNA) transfection and subsequent immortalization of human prostate epithelial cells. METHODS: Recently, two human prostate epithelial cell lines were established by HPV transformation: PZ-HPV-7, derived from normal peripheral zone (PZ) tissue, and CA-HPV-10, derived from high Gleason grade adenocarcinoma. Expression of PSA was studied by the reverse transcription polymerase chain reaction (RT-PCR), because in preliminary studies using immunocytochemistry and Northern blotting, no PSA expression was found. PSM was analyzed by RT-PCR and nested RT-PCR. These analyses included primary human prostate cell strains. Furthermore, androgen-supplemented methylthiazol tetrazolium (MTT) growth assays were performed and expression of AR was studied by immunocytochemistry. Prostate carcinoma cell lines LNCaP and PC-346C were included as positive controls and breast carcinoma cell line MCF-7 as a negative control. RESULTS: Both cell lines exhibited low levels of RNA for PSA and PSM in comparison with cell lines LNCaP and PC-346C. AR expression by immunocytochemistry was negative using monoclonal antibody F39.4 and polyclonal antibody SP-197. In an androgen-supplemented environment, growth rates of both HPV immortalized cell lines were not stimulated in contrast to LNCaP. CONCLUSIONS: RNA transcripts of PSA and PSM were detected by RT-PCR in HPV immortalized prostate epithelial cell lines PZ-HPV-7 and CA-HPV-10. The expression of prostate-specific markers may further validate the utility of this stepwise transformation model of human prostate carcinogenesis.
Assuntos
Antígenos de Superfície/biossíntese , Carboxipeptidases/biossíntese , Papillomaviridae/genética , Antígeno Prostático Específico/biossíntese , Próstata/metabolismo , Receptores Androgênicos/biossíntese , Transfecção , Antígenos de Superfície/análise , Carboxipeptidases/análise , Células Cultivadas , Glutamato Carboxipeptidase II , Humanos , Masculino , Reação em Cadeia da Polimerase , Próstata/citologia , Antígeno Prostático Específico/análise , Receptores Androgênicos/análiseRESUMO
The pharmacokinetics of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, were investigated in BALB/c (nude) mice after i.p. injection and after oral administration of radiolabeled drug. After i.p. injection the compound was rapidly cleared from the serum (t1/2 alpha = 14 min; t1/2 beta = 2.1 h) and from tissues such as muscle, liver and kidney (t1/2 alpha = 30-60 min; t1/2 beta = 2.1 h). DFMO concentrations were proportional to the administered dose (10-2000 mg/kg) in both serum and tissues. Oral administration of DFMO was carried out by dissolving the compound in drinking water at a concentration of 20 g/l. Studies on the distribution showed that DFMO did not accumulate preferentially in any particular tissue. An extremely wide variation in the dose actually achieved in different animals was observed; this ranged from 350 to 2800 mg/kg for a 14-h treatment period. A significant correlation (r = 0.83-0.92) between the dose of DFMO, calculated from the consumption of drinking water for each individual animal, and the DFMO concentrations in serum, muscle, spleen, liver and kidney was found. Similarly, it was shown that oral administration of DFMO during the daytime resulted in 10- to 15-fold lower levels than administration during the night. After discontinuation of treatment DFMO levels in serum and tissues decreased by 50% in approximately 6 h. From these results it is concluded that the optimal treatment schedule of mice with DFMO (or other drugs with similar pharmacodynamic properties) consists in a combination of oral administration via the drinking water and additional i.p. injection (during the daytime). Furthermore, the drug intake of the individual animals should be monitored to check whether the experimental requirements are actually fulfilled.