The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Abstract

Prothymosin alpha (proTalpha) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTalpha is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTalpha presenting maximum immunomodulatory activity. Calf thymus proTalpha was trypsinised, and the five fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTalpha(89-102) and proTalpha(103-109) significantly fortified healthy donor-lymphocytes' immune responses to levels comparable to those induced by intact proTalpha. These effects were more pronounced in cancer patients, where peptides proTalpha(89-102) and proTalpha(103-109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTalpha. ProTalpha(1-14), proTalpha(21-30) and proTalpha(31-87) marginally upregulated lymphocyte activation. This is the first report showing that proTalpha's immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTalpha fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy

“…ProT (100)(101)(102)(103)(104)(105)(106)(107)(108)(109) induces PBMC proliferation and cytotoxicity, promotes the maturation of dendritic cells (DC), adopts a -sheet conformation, and its eVects are sequence-speciWc and comparable to that of intact proT . In an earlier study, we had shown that a slightly smaller segment (103)(104)(105)(106)(107)(108)(109) was also eVective in restoring the immune function of PBMC obtained from cancer patients in vitro [90]. As for today, the peptide TKKQKTDEDD is known to be generated in vivo upon caspase-cleavage of proT during apoptosis [91,92], and additional mechanisms of its generation and immunoreactivity are currently being investigated in our laboratory.…”

Prothymosin α and a prothymosin α-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

“…T2 cells were incubated for 2 h at 37°C with 10 μg/mL HER-2(9 369 ) or tyr(9 369 ), washed and labeled with sodium chromate, as previously described [21]. Non-loaded T2 were similarly labeled for controls.…”

“…We and others have previously shown that proTα upregulates the expression of IRAK-4 in human monocytes [19], ligates TLR-4 on murine macrophages and signals through MyD88-dependent and independent pathways [20]. Similar to its immunoreactive decapeptide proTα(100–109) [21], it upregulates the expression of HLA-DR [22], CD80, CD83 and CD86 and promotes maturation of human DCs in vitro [23]. …”

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

“…Although its biological role has not been completely elucidated, literature points toward a dual role for ProTa: an extracellular one, associated with cell-mediated immunity (Cordero et al 1997;Skopeliti et al 2006), and an intracellular one, related to cell proliferation and apoptosis (Bustelo et al 1991;Rodriguez et al 1998;Jiang et al 2003). Increased intracellular expression of ProTa, which has been thought to be an oncoprotein (Karapetian et al 2005;Kobayashi et al 2006), has been observed in several types of human cancer.…”