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Concurrent radiotherapy and chemotherapy result in a significant benefit with respect to induction chemotherapy followed by radiotherapy or radiotherapy alone, although with a significant increase of toxicity. To discover a more tolerated and effective chemoradiation regimen, the feasibility and efficacy of a hyperfractionated accelerated irradiation with concurrent protracted venous infusion chemotherapy was investigated. Sixty-five patients with advanced head and neck cancer underwent a definitive (53 patients) or a postoperative adjuvant (12 patients) chemoradiation treatment. Chemotherapy consisted of an intravenous protracted infusion of 5 and 200 mg/m /d cisplatin and 5-fluorouracil, respectively. Radiotherapy consisted of a split-course accelerated hyperfractionation of two 150-cGy (split twice a day) or three 100-cGy fractions per day (split three times a day) at more than 6-hour intervals, for 2 weeks followed, after a 1-week interruption, by 2-to-3-week treatment, with the same fractionation schedule, to a total dose of 60 Gy to 69 Gy. Confluent mucositis was tolerable and was the cause of treatment delay of more than 10 days in only 20% of patients. Grade 3 or greater systemic toxicity occurred only in 9 of 65 (14%) patients and was never the cause of drug dose reduction. Complete responses were observed in 69% of patients with gross diseases. At a median follow-up of 43.5 months, 45% of patients were alive and free of disease and 38% died of cancer. The 5-year actuarial local regional failure was 35%. The 5-year actuarial disease-specific survival was 50%. Preservation of larynx function was achieved in 47% of living patients and in 74% of all patients, with advanced tumors of the laryngopharynx. The long-term results of this study suggest that this chemoradiation regimen has the potential of achieving a significant improvement over standard therapy while avoiding significant toxicity

We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central alpha-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.

We have studied the relationship between the occurrence of chromatin diminution and the developmental behavior of somatic blastomeres in early development of the nematode Parascaris univalens. A cytological and immunocytochemical analysis of chromatin diminution was performed in P. univalens embryos exposed to ‘vegetalizing’ (LiCl) and ‘animalizing’ (NaSCN) substances during early developmental stages. We have also analyzed chromatin diminution in embryos displaying only symmetrical ‘somatic-like’ divisions due to a brief cytochalasin B treatment at the pronuclear stage. The results show that LiCl treatment induces chromatin diminution in P0-P4 pregerminal blastomeres while NaSCN treatment prevents it. Pregerminal cells undergoing chromatin diminution in ‘vegetalized’ embryos behaved like somatic cells with respect to division and cleavage patterns. NaSCN treatment results in undiminuted polynucleated embryos that are not capable of cleavage. In cytochalasin B-pulsed embryos, chromatin diminution occurs in all blastomeres. From our results we conclude that chromatin diminution and somatic cell behavior are not separable and constitute strictly correlated events in Parascaris. Moreover, the results indicate that the segregation of the cytoplasmic factors involved in chromatin diminution in early Parascaris development are microfilament-mediated. Here, we also report the formation of a latter pregerminal cell precursor (P5) not susceptible to LiCl-induced chromatin diminution.

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.