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Clusterin/Apolipoprotein J (CLU) is a cellular senescence biomarker implicated in several physiological processes. In this work we have investigated CLU expression and function in human haematopoietic cells. We found that early passage human T cell clones (TCC) express minimal endogenous amounts of CLU, which are significantly elevated in late passage cells. Moreover, exposure of TCC to increased levels of the essential micronutrient zinc in culture resulted in intense induction of CLU. Because haematopoietic cells cease proliferation following induction of terminal differentiation, we also studied the expression profile of CLU in the leukemic progenitor cell lines K562 and HL-60. We found that, like TCC, both cell lines express minimal endogenous levels of CLU in their actively proliferating state. However, when induced to differentiate into their distinct cell types, CLU was found to be up-regulated specifically in those cells expressing the main differentiation markers. Enforced stable over-expression of CLU in K562 cells inhibited the expression of the CD14 differentiation marker and blocked differentiation to either monocytes/megacaryoblasts or to erythrocytes. Overall, our results suggest that CLU is actively involved in both replicative senescence and terminal differentiation in different types of human haematopoietic cells.

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR‐Dextran, using Immunofect MG beads, indicates that transient holes of 5.4 nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15–30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

Previous work from this laboratory has shown that immunoporation has the potential for the selective transfection of a range of different animal cells based on their immunological identity. The unique ability of immunoporation to target cells for transfection combined with the high efficiency of transfection and the high viability of cells make this method extremely promising for scientific and medical research. The experiments reported here show that not only can immunoporation produce transient transfection but also stably transfected cells are produced and such types of cells will be essential for the use of this method for gene therapy.

Immunoporation is a novel method of cell transfection based upon the use of a new type of beads, Immunofect beads, that can be targeted to make holes in different types of cells depending on the type of bead used. It is known that the efficiency of transfection of cells by some techniques can be affected by the presence of serum and another important factor that appears to affect transfection efficiency and cell viability is the osmolarity of the transfection medium. This report presents studies on the effects of serum and varying osmolarity on the efficiency of transfection using immunoporation. The results clearly indicate that in hypertonic media the presence of serum decreases the efficiency of transfection. In the case of osmolarity, increasing the osmolarity of the immunoporation medium increases the efficiency of transfection but above about 650 mOsm this increasing efficiency is offset by the much lower viability of the cells.

Cell transfection is now a central technique in molecular biology and an essential prerequisite for gene therapy. Here we describe how beads coated with antibodies and bound to specific cell-surface transmembrane proteins can create holes in cells when the beads are removed, allowing transfection of the cells with DNA or other macromolecules. This unique targeted transfection of cells by immunoporation is very efficient and results in minimal cell death.