The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.

Anorexia and weight loss are serious complications that adversely effect the prognosis of cancer patients. It has been suggested that TNF/cachectin may cause cachexia. To determine If TNF/cachectin can induce progressive weight loss in tumor-bearing animals, a clone of the human TNF/cachectin gene was isolated and inserted into a mammalian expression vector. This construct was transfected into CHO cells, and a cell line (CHOTNF-20) that secretes TNF/cachectin was isolated. A cell line (CHOCMV-Neo) that contains the same expression vector without the TNF/cachectin gene was also isolated. Nude mice injected intra-peritoneally with CHOTNF-20 cells died more quickly than mice injected with CHOCMV-Neo cells. Eighty-seven percent of mice inoculated intramuscularly with CHOTNF-20 cells developed severe cachexia and weight loss. All mice bearing CHOCMV-Neo tumors maintained or increased their body weight. We conclude that mice bearing tumors that secrete TNF/cachectin develop progressive wasting and die more quickly than mice bearing control tumors.