p53 gene transfer does not enhance E2F-1-mediated apoptosis in human colon cancer cells

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.

p53 gene transfer does not enhance E2F-1-mediated apoptosis in human colon cancer cells

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.

THE ORIGINS OF THE BRAIN STEM PROJECTIONS TO THE SPINAL CORD WITH THE RETROGRADE METHOD OF HORSERADISH PEROXIDES (HRP) / 解剖学报

The efferent projections to the spinal cord from the brain stem were studied with HRP. Fifty per cent solution of HRP was slowly injected into the intumescentia cervicalis at six points on its right side in 10 albino rats and the HRP-labelled cells were found in the following nuclei:1. Of the midbrain: the nucleus linearis, nucleus raphe dorsalis, substantia nigra, nucleus ruber, nucleus cuneiformis, nucleus colliculus superior, nucleus Edinger-Westphal, nucleus Darkschewitsch, nucleus interstitialis (Cajal), nucleus ventralis tegmenti and nucleus dorsalis tegmentalis;2. Of the ports: the nucleus raphe magnus, nucleus medianus raphe, nucleus trigemini principalis, nucleus lemnisci lateralis, nucleus reticularis pontis oralis, nucleus reticularis pontis caudalis, locus ceruleus, nucleus subceruleus, nucleus vestibularis lateralis, nucleus vestibularis medialis, nucleus parabrachialis and nucleus olivaris su- perior;3. Of the medulla oblongata: the nucleus raphe obscurus, nucleus raphe pallidus, nucleus gracilis, nucleus cuneatus, nucleus tractus spinalis nervi trigemini, nucleus tractus solitarius, nucleus commissuralis (Cajal), nucleus reticularis lateralis, nucleus reticularis paramedianus, nucleus reticularis gigantocellularis and nucleus olivaris inferior;4. Of the cerebellum: the nucleus medialis and lateralis.The functions of spinal projection from the nuclei raphe, reticularis, locus ceruleus, substantia nigra and the nucleus dorsalis tegmentalis have been discussed.

THE NEURONAL CONNECTIONS OF THE PARAFASCICULAR NUCLEUS, SUBPARAFASCICULAR NUCLEUS AND VENTROMEDIAL NUCLEUS OF THE THALAMUS WITH THE SPINAL CORD / 解剖学报

Attempts were made to determine the cells of origin from the thalamus to the spinal cord by using the retrograde tracer technique with horseradish peroxidase (HRP). The solution of HRP was injected into the intumescentia cervicalis unilaterally in eight albino rats. The results indicated that the HRP-labeled cells were located in the parafascicular nucleus, subparafascicular nucleus and the ventromedial nucleus of the thalamus. The function of spinal projection from these neuclei were discussed.

THE ORIGIN OF THE HYPOTHALAMIC PROJECTION TO THE SPINAL CORD——HRP METHOD / 解剖学报

Seven adult albino rats were used for this study. The 50％ HRP solution was injected into the cervical enlargment unilaterally. Labelled neurons were mainly found in the nucleus paraventricularis, nucleus lateralis, nucleus paraventricularis, nucleus lateralis, nucleus perifornicalis, nucleus hypothalamicus anterior, nucleus hypothalamus posterior, nucleus suprachiasmaticus, nucleus supraopticus and nucleus periventricularis ipsilaterally. Most of the labelled neurons were found in nucleus paraventricularis hypothalamicus, less in the nucleus lateralis and only a few in other nuclei.The authors suggest that the efferent projections from the hypothalamus to the spinal cord in the rat may play a role in the integrative function of the spinal cord. which may be involved in the process of acupuncture analgesia.