ABSTRACT. Lymphoproliferative disorders associated with Epstein- Barr virus (EBV) infections can occur in the setting of immunosuppression. In some patients, the lymphoproliferative disorder can resemble an aggressive monoclonal non-Hodgkins lymphoma (NHL). These NHL are poorly responsive to conventional therapy. Similarly, antiviral therapy with synthetic nucleosides such as ganciclovir are ineffective because the genes that render the virus susceptible to therapy are not expressed in EBV+ lymphomas. Using a cell line derived from a lung transplant recipient with an EBV+ immunoblastic NHL, we studied the ability of arginine butyrate to induce the expression of EBV thymidine kinase. Arginine butyrate was not only effective in inducing EBV thymidine kinase transcription, but also acted synergistically with the antiviral agent ganciclovir to inhibit cell proliferation and decrease cell viability. Based on these findings, the patient from whom the cell line was derived was treated with arginine butyrate/ganciclovir as well as conventional cytotoxic chemotherapy. No additional toxicity was observed with the arginine butyrate/ganciclovir therapy. Histologic examination of the tumor showed substantial necrosis. These observations suggest the feasibility of arginine butyrate induction of ganciclovir susceptibility in patients with EBV- associated lymphomas.

Keywords: Immunosuppression, lymphoproliferative disease; post- transplant, lung transplantation, antiviral, thymidine kinase.

Reprint requests to: Steven J. Mentzer, M.D., Division of Thoracic Surgery, Brigham & Women's Hospital, 75 Francis Street, Boston MA 02115.

ABSTRACT: To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34+++ cells which are highly enriched for hematopoietic stem / progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H- ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34+++ cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Keywords: H-ras, erythroid differentiation, TF1 cells, CD34+++ cells, cord blood.

Reprint requests to: Li Lu, M.D., Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut Street, Room 302, Indianapolis, IN 46202, phone: (317) 274-7560, fax; (317) 274-7592, e-mail: llu@iupui.edu.

Communicated on April 28, 1998, by John Yu, M.D.,Ph.D., The Scripps Research Institute, La Jolla, California.

ABSTRACT. p21cip1/waf1 is a cyclin dependent kinase inhibitor. We have previously reported stimulation of p21cip1/waf1 by steel factor and GM-CSF in a factor dependent cell line and of p21cip1/waf1 involvement in hematopoiesis in vivo in p21cip1/waf1 gene knockout (-/-) mice. To further assess a role for increased p21cip1/waf1 in hematopoietic progenitor cells, we developed the retroviral vector L(p21cip1)SN to transcriptionally regulate p21cip1/waf1 from the Mo-MLV LTR. L(p21cip1)SN and the control vector LXSN were used to transduce murine bone marrow progenitor cells from p21cip1/waf1 (-/-) and littermate control (+/+) mice, as well as from other mouse strains. Hematopoietic colony formation by transduced cells was assessed in semi-solid culture medium with multiple growth factors. Myeloid colony formation by bone marrow cells from p21cip1/waf1 (-/-) mice was significantly lower than that by (+/+) mouse cells. Transduction of cells with LXSN had no effect on colony formation; however, (-/-) cells transduced with L(p21cip1)SN formed significantly greater numbers of colonies than either LXSN-transduced (-/-) or (+/+) cells. Moreover, L(p21cip1)SN-transduced (+/+) cells formed significantly more colonies than LXSN-transduced (+/+) cells. Increased cloning efficiency of progenitors from normal strains of mice induced by L(p21cip1)SN compared to LXSN-transduced cells was seen whether unseparated or highly purified populations of Sca1+ Lin- marrow cells were used. Gene transfer of L(p21cip1)SN increased the size and number of cells per colony, as well as the number of colonies compared to LXSN gene transfer. No colonies grew from non-transduced, LXSN-, or L(p21cip1)SN-transduced cells when no growth factors were added to the cultures. These results document the positive effect of p21cip1/waf1 in the proliferation and/or differentiation of the murine myeloid progenitor cells that lead to colony formation.

Keywords: p21cip1/waf1, gene transfer, p21cip1/waf1 knockout mice,hematopoietic progenitor cells.

Reprint requests to: Stephen E. Braun, Ph.D., Walther Oncology Center, 1044 W. Walnut Street, Room 302 R4, Indianapolis, IN 46202-5121, phone: (317) 274-7555, fax: (317) 274-7592, e-mail: sbraun@iupui.edu.

ABSTRACT. Significant amounts of ribonuclease inhibitor protein are present in human and rat erythrocytes, cells that are essentially devoid of ribonuclease or functional RNA. The protein from human erythrocytes is indistinguishable from human placental ribonuclease inhibitor protein by immunological and biochemical criteria. Each inhibitor forms an equimolar complex with bovine pancreatic ribonuclease A and is inactivated by treatment with the sulfhydryl reagent p-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identity of the erythrocyte and placental proteins. We calculate a level of 1.5 - 3.5 x 10(4) molecules of active inhibitor per erythrocyte, most or all of which occurs in an uncomplexed form since inactivation of the inhibitor revealed barely detectable levels of RNase activity. Immunogold localization showed a high level of labeling and a uniform distribution of gold particles in the cytoplasm of erythrocytes, while little inhibitor activity was found in association with isolated red blood cell membranes. Oxidative stress on isolated red cells resulted in a decrease in the level of reduced glutathione and a gradual and irreversible loss of inhibitor activity; inhibitor disappeared from the cytosol and became associated with nascent Heinz bodies. We suggest a role for this protein in the metabolism and aging process of the erythrocyte.

Keywords: Red blood cell, RNASIN, ribonuclease inhibitor, Heinz bodies, oxidative stress, aging.

Reprint requests to: Dohn Glitz, Ph.D., Department of Biological Chemistry, UCLA School of Medicine, P.O. Box 951737, Los Angeles, CA 90095-1737, phone: (310) 825-1144, fax: (310) 206-5272, e-mail: Dglitz@biochem.medsch.ucla.edu.