L-428

L428 Cells Produce E Rosette-Inhibiting Factor

The diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. A comparison of our Hodgkin cell lines showed not only the highest activity in fresh L428 supernatant but also the highest durability. The effect of serial dilution of rosette inhibiting factor (RIF) activity is shown in Fig. 1. At high- or low-factor concentration, no effect on E rosetting could be detected, whereas in between, significant inhibition was observed.

The half-life of the RIF activity in crude supernatant or partially purified material in solution at 4°C or -20°C was about 3 weeks. RIF was stable over months if stored at -80°C or as a lyophilized preparation at -20°C. RIF was inactivated under mild acidic (pH 6) or alkaline (pH 8) conditions (Table 1). RIF activity of partially purified supernatant (after ion exchange chromatography) was retained when treated with neuraminidase but did not withstand trypsin incubation.

Purification of lyophilized active fractions was performed either on a hydroxylapatite column (elution of active RIF at 0.2 mol/L and between 0.4 and 0.6 mol/L phosphate concentration) or anion exchange column (elution of RIF activity at a NaCl concentration between 0.38 and 0.44 mol/L). Sequential purification by molecular sieve and anion exchange chromatography yielded pure protein, as documented by SDS-PAGE (Fig. 2) with RIF activity. Aminoacid analysis of the 12.5-Kd and 50-Kd protein bands gave identical results.

Fig. 1 Dose-response relationship of RIF activity. (Katay I, et al., 1990)

Table 1. Chemical properties of RIF. (Katay I, et al., 1990)

Fig. 2 SDS-PAGE. (Katay I, et al., 1990)

Melittin Increases Cisplatin Sensitivity and Kills L-428 Cells

HL is a neoplasia with high cure rates. Melittin (MEL) is a pore-forming peptide from bee venom. Anti-cancer activities of MEL have been proposed 50 years ago. At the same time, MEL has been described as a putative tumor promoter with similar effects as phorbol esters.

KM-H2 or L-428 cells were co-cultured with normal peripheral blood mononuclear cells (PBMC) in the presence of different concentrations of MEL. As shown in Fig. 3, the percentage of living tumor cells decreased with increasing MEL concentrations. L-428 cells were pre-incubated with a low concentration of MEL and tested sensitivity for cisplatin. As shown in Fig. 4, MEL pre-treated L-428 cells showed significantly enhanced sensitivity for cisplatin. In KM-H2 cells, which have a higher basal sensitivity for cisplatin, only marginal effects were observed (Fig. 4). No increased Rh123 efflux in L-428 cells after treatment with MEL (Fig. 5), suggesting that ABCB1 transporter activity plays no major role in the drug sensitivity of these cells.

Fig. 3 Toxicity of MEL for HL cell lines KM-H2 and L-428. (Kreinest T, et al., 2020)

Fig. 4 MEL increases cisplatin sensitivity of resistant L-428 cells. (Kreinest T, et al., 2020)

Fig. 5 MEL has no effect on ABC transporter activity in L-428 cells. (Kreinest T, et al., 2020)