Francoise Geisen

Inhibitory effects of the nucleoside analogue gemcitabine on prostatic carcinoma cells

Gemcitabine (2≺,2≺difluoro‐2≺deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen‐sensitive tumor cell line LNCaP and the androgen‐insensitive cell lines PC‐3 and DU‐145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co‐administration of 10–100 μM of its natural analogue deoxycytidine. In view of a future clinical application of this anti‐tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic‐macrophagic progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic‐macrophagic progenitor cells required a concentration of 9 nM. Co‐administration of deoxycytidine to gemcitabine‐treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic‐macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma.

In vivo activation of circulating monocytes by exogenous growth hormone in man

Animal studies have shown that administration of growth hormone improves wound healing. Monocyte activation is a prerequisite for optimal repair of damage. In vitro, human recombinant growth hormone was shown to be a potent human monocyte chemoattractant. It induced random migration and chemotaxis at picomolar concentrations of recombinant human growth hormone; combinations of growth hormone with other chemoattractants deactivated the chemotactic response. Other functions of monocytes that are activated by growth hormone include release of superoxide anion and production of cytokines. In order to test activation of human monocytes by growth hormone in vivo, we investigated the effects of recombinant human growth hormone administration on monocyte migration in nine healthy young adults. After a single dose of recombinant human growth hormone (4 IU subcutaneously injected), random migration of circulating monocytes significantly increased, whereas chemotaxis of monocytes that was maximally stimulated with f-Meth-Leu-Phe decreased (p < .05). The alterations paralleled the concomitantly measured plasma levels of growth hormone. After recombinant human growth hormone administration, no changes were seen in plasma levels of proinflammatory cytokines. These in vivo data on monocyte migration are comparable to effects of growth hormone on monocyte migration in vitro and strongly suggest that recombinant human growth hormone can activate circulating monocytes in man.

2′,2′-Difluorodeoxycytidine (Gemcitabine) Induces Apoptosis in Myeloma Cell Lines Resistant to Steroids and 2-Chlorodeoxyadenosine (2-CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2‐chlorodeoxyadenosine (2‐CdA, cladribine) and the pyrimidine antagonist 2′,2′‐difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10−6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 μM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 μM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2‐CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells.

T Cells and Natural Killer Cells after Treatment of Hairy Cell Leukaemia with 2-Chlorodeoxyadenosine

We report that continuous suppression of CD4+ lymphocyte subsets does not necessarily lead to an increased rate of infections more than 6 months after treatment of hairy cell leukemia (HCL) with 2-chlorodeoxyadenosine (cladribine; 2-CdA). In a retrospective analysis of 17 consecutive patients with HCL treated with 2-CdA and followed thereafter for an average of 440 days (ranging from 71 to 1,046 days), all lymphocyte subsets significantly decreased after therapy. However, natural killer (NK) cells increased to 203 cells/microliter during the following 4 months, whereas CD3+ and CD4+ T cell subsets did not reach pretreatment values even more than 1 year after 2-CdA therapy. In our study group, 5 opportunistic infections occurred during the first 2 weeks after treatment with 2-CdA, and no severe infections were registered after this early leukopenic phase. A review of the current literature revealed that at least 56 infections occurred in 158 patients repeatedly treated with 2CdA for low-grade lymphoproliferative diseases other than HCL (= 35%). In patients with HCL, the rapid increase of NK cell counts after 6 months might be responsible for reducing the risk of severe infections after the early leukopenic phase despite CD4+ cell suppression.

Differential effects of cladribine and gemcitabine on erythroid and granulocytic progenitors from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal neoplastic disease that originates in a pluripotent stem cell. Selection of normal progenitors by graft-purging may improve the outcome after autologous transplantation. In our methylcellulose assays, the nucleoside analogs cladribine (2-CdA) and gemcitabine (dFdC) showed more prominent inhibitory effects on CML than normal bone marrow (BM) progenitors. For dFdC, however, long-term incubations were necessary to achieve complete inhibition. Deoxycytidine kinase, the key enzyme of both 2-CdA and dFdC metabolisms, was only partially responsible for this differential sensitivity. We suggest that 2-CdA and dFdC might be helpful in purging of CML BM cells before autologous BM transplantation. Further studies on more primitive cells are warranted.

Local application of antimycotics in mucormycosis cerebri: a case report.

A 65 year old man was found to have mucormycosis cerebri during immunosuppression after treatment of hairy cell leukaemia with 2-chlorodeoxyadenosine. Although mucormycosis cerebri has a poor prognosis, the patient survived after systemic administration of high dose amphotericin B, extensive excision of the abscess, and additional local application of amphotericin B with the help of an absorbable gelatin sponge.

THE IMMUNOSUPPRESSIVE SUBSTANCE 2-CHLORO-2-DEOXYADENOSINE MODULATES LIPOPROTEIN METABOLISM IN A MURINE MACROPHAGE CELL LINE (P388 CELLS)

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5–20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [14C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P<0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [14C]OA into the CE fraction than in the presence of 2-CdA alone (P<0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation. However, incubation of P388 cells with 20 nM 2-CdA did not result in a decrease in cellular NAD content. As 20 nM 2-CdA showed no effect on intracellular cholesterol synthesis based on measurement by 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, the decrease in cellular cholesterol content and in [14C]OA incorporation seems to be primarily due to an interference with Ac-LDL metabolism.

New Rearrangement Pattern after Treatment of Hairy-Cell Leukemia with 2-Chlorodeoxyadenosine

Leukemic hairy cells are clonally proliferating B-lymphoid cells with clonal rearrangements of genes for immunoglobulin chains. We describe a patient with a new hairy-cell clone after treatment with 2-chlorodeoxyadenosine (2-CdA). In this patient, a single course of 2-CdA resulted in good partial remission of hairy-cell leukemia, but Southern blot analysis of bone marrow biopsies and polymerase chain reaction using seminested amplifications with consensus primers revealed a new rearranged band 4 months after therapy with 2-CdA. Four years after therapy, the patient is in complete clinical remission and both bands disappeared during follow-up. The new rearranged band might have been related to prior treatment of hairy-cell leukemia with 2-CdA.