Establishment of tumor cell lines as an independent prognostic factor for survival time in patients with small-cell lung cancer.

We studied tumor samples from 39 patients, who entered our study from January 1989 to May 1990, to assess whether the ability to establish a continually growing tumor cell line from fresh tumor specimens can be associated with decreased survival times in patients with small-cell lung cancer. The tumor samples were used to establish cell lines in culture using a serum-free medium supplemented with hydrocortisone, insulin, transferrin, estrogen, and selenium (HITES). Thirty-three of these specimens were obtained by fiberoptic bronchoscopy from primary sites during routine diagnostic procedures. A total of 11 (28%) cell lines were established: seven (21%) from 33 primary tumors and four (80%) from five peripheral lymph nodes. Survival times of the 11 patients whose tumor cell specimens continually grew in culture at any time during their clinical course were significantly shorter than those of the 28 patients whose tumor cell specimens did not grow in vitro (median survival time of 26 weeks versus 73 weeks; P = .0068). Cox's proportional hazards model, including sex, age, Eastern Cooperative Oncology Group performance status, stage, source of specimen, treatment, and in vitro tumor cell growth in the overall patient group, showed that cell line establishment (P = .0017) and no therapy (P = .0015) were the most important factors indicating poor survival time. For the subgroup of 23 primary tumor patients, the important factors (in decreasing order) that indicated decreased survival times were the establishment of a cell line (P = .0112) and with cyclophosphamide-doxorubicin-vincristine alternating with cisplatin-etoposide, versus cisplatin-vincristine-doxorubicin-etoposide therapy (P = .0463). Our study demonstrates that in vitro tumor cell growth is an adverse predominant prognostic factor in patients with small-cell lung cancer.

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme.

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.

Occurrence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation.

Specification of the dorsoventral axis is a subject of great importance in amphibian embryogenesis. We have found that cytoplasm of the vegetal dorsal cells of a 16-cell embryo of Xenopus laevis, when injected into the ventral vegetal cells of a recipient at the same stage, can induce formation of a second axis. In the present experiments,using the same assay procedure, we found that the cytoplasm around the vegetal pole of an egg before cortical rotation is also active in inducing a second axis,that the activity decreases throughout the second half of the cell cycle and appears in a presumptive dorsal equatorial region at the 2- to 16-cell stages. This is the first demonstration of the localization of dorsal forming activity in any specific region of an egg. After UV irradiation,a treatment that is known to block cortical rotation and thereby inhibit axis specification, the activity remains near the vegetal pole beyond the first cell cycle and does not appear in an equatorial region, at least at the 16-cell stage. This suggests that cortical rotation ora related force is in some way involved in changes in distribution of the activity.We also found that UV-irradiated 8-cell embryos can rescue dorsal development when they are cut into halves along the first cleavage plane. Histological examination revealed that the rescued embryos have a neural tube and notochord. In the half embryo, the animal and vegetal regions came into contact during wound healing, an event that enables the activity to localize in the new equator of an embryo. Therefore this rescue suggests that, if the activity is distributed only in the equatorial region, dorsal specification occurs. In fact, the dorsal side of the rescued embryos seems to correspond to the plane through which the embryos have been cut.Based on our results, we propose (1) that a determinant that carries axis-inducing activity is first present around the vegetal pole, (2) that the determinant shifts from the vegetal pole to an equatorial region by or in close association with cortical rotation and (3) that occurrence of the determinant in the equatorial region is a prerequisite for axis specification.

A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis.

In Xenopus laevis, dorsal cells that arise at the future dorsal side of an early cleaving embryo have already acquired the ability to cause axis formation. Since the distribution of cytoplasmic components is markedly heterogeneous in an egg and embryo, it has been supposed that the dorsal cells are endowed with the activity to form axial structures by inheriting a unique cytoplasmic component or components localized in the dorsal region of an egg or embryo. However, there has been no direct evidence for this. To examine the activity of the cytoplasm of dorsal cells, we injected cytoplasm (dorsal cytoplasm) from dorsal vegetal cells of a Xenopus 16-cell embryo into ventral vegetal cells of a simultaneous recipient. The cytoplasm caused secondary axis formation in 42% of recipients. Histological examination revealed that well-developed secondary axes included notochord, as well as a neural tube and somites. However, injection of cytoplasm of ventral vegetal cells never caused secondary axis and most recipients became normal tailbud embryos. Furthermore, about two-thirds of ventral isolated halves injected with dorsal cytoplasm formed axial structures. These results show that dorsal, but not ventral, cytoplasm contains the component or components responsible for axis formation. This can be the first step towards identifying the molecular basis of dorsal axis formation.