Kinetic analysis of human IL-2 activated cytotoxic cells.

Kinetic analysis was used to define lytic events in peripheral blood mononuclear cells (PBMC) activated in lymphokine conditioned medium (LCM) and recombinant interleukin-2 (rIL-2). This analysis provided quantitative information on the functional properties of these lymphokine-activated killer (LAK) cells against NK-resistant and NK-sensitive tumor cell lines. The maximum rate of target cell lysis (Vmax) and Km (target cell number resulting in 1/2 Vmax) were determined. IL-2 activated effector cells that bound to target cells also lysed them (i.e., non-lytic bystander lymphocytes did not influence the determination of kinetic parameters) in contrast to lysis mediated by unactivated NK cells. The extent of LAK cell binding to tumor target cells was dependent upon the tumor type. LAK cell frequency determinations were calculated where Km approximated the concentration of LAK cells that were capable of killing a particular target. LAK cells generated in rIL-2 were lytically more efficient than those activated in LCM, and T-depletion resulted in a LAK population with the highest maximum rate of lysis. The use of kinetic analysis to evaluate LAK cell frequencies and quantitate lytic events will be useful in determining the effects of drugs, biological response modifiers and disease states on LAK cell function.

Ability of circular extrachromosomal DNA molecules to carry amplified MYCN proto-oncogenes in human neuroblastomas in vivo.

Amplification of the proto-oncogene MYCN (also known as N-myc) in neuroblastomas has been shown to correlate with both disease stage and prognosis, yet little is known about the DNA structures that carry amplified MYCN genes in neuroblastomas in vivo. We have used DNA irradiation and pulsed-field gel electrophoresis to analyze MYCN amplification structures in eight neuroblastomas from separate patients (four primary tumors and four metastatic lesions exhibiting MYCN amplification). Six of the eight neuroblastomas (three primary tumors and three metastatic lesions) exhibited MYCN DNA irradiation profiles consistent with the presence of circular extrachromosomal DNA amplification structures. Five neuroblastomas possessed amplification structures within the size range of double minute chromosomes, and one contained smaller DNA circles. Two neuroblastomas exhibited MYCN DNA irradiation patterns consistent with larger (presumably chromosomal) amplification structures. Multiple sizes of DNA circles were observed in the neuroblastomas of four different patients, implying in vivo multimerization of amplification structures. The presence of circular MYCN amplification structures in six of eight neuroblastomas examined suggests that circular DNA molecules are important structures in in vivo gene amplification.

In vitro enhancement of peripheral blood mononuclear cell natural killer activity following short term incubation with fetal calf serum.

This study demonstrates that short term incubation of blood mononuclear cells (MC) in heterologous sera enhances their natural killer (NK) but not their antibody dependent killer (K) activity, and confirms that NK stimulation is not related to blastogenesis. MC were obtained from healthy donors and incubated with RPMI 1640 supplemented with various serum sources. NK and K activity of incubated vs. fresh MC against SK-N-SH, Chang or Raji cell line targets were compared in a 4-hr 51Cr release assay. A significant (p less 0.01) increase in NK cytotoxicity was detected when MC were incubated with fetal calf serum (FCS) or human AB serum (ABS) at 37 degrees C. When a more sensitive NK target cell (K-562) was used only the cells incubated in FCS demonstrated increased NK cytotoxicity. Augmentation of NK cell activity by FCS was not related to blastogenesis, mitosis, natural antibodies against lymphocytes or target cells, immunoglobulin complexes or alterations in MC OKT4 and OKT8 subpopulations. In contrast to NK activity, K cytotoxicity was not increased after incubation at 37 degrees C in FCS or ABS, and it was depressed in IPT (p less than 0.05). Thus FCS is capable of stimulating NK cell activity against human tumor cell lines in less in less than 24 hr.