A p53 growth arrest protects fibroblasts from anticancer agents.

Reversible inhibitors of the cell cycle such as the TGF-betas have been exploited to protect dividing cells from exposure to anticancer drugs and radiation. Here, rat embryo fibroblast (REF) lines expressing different p53 mutations were used to test whether the p53 growth arrest could also chemoprotect cells from high doses of anticancer drugs. Whereas the doubling times of the different REF lines at 37 degrees C were similar, cells bearing temperature-sensitive mutations (mouse 135V or human 143A) were growth arrested at 31 degrees C. Temperature-dependent p53 activity was associated with increased levels of MDM2 and p21/WAF1, and the induction of an integrated p53-responsive luciferase gene. The REF lines exhibited similar sensitivities to common anticancer drugs when grown at 37 degrees C. However, when exposed to the same agents following transient incubation at 31 degrees C, the p53-arrested cells exhibited a marked survival advantage as shown by colony-forming assays. Chemoprotection was not universal, in that colony formation was not enhanced significantly after treatment with cisplatin or 5-fluorouracil, two drugs which can cause cellular damage throughout the cell cycle. Like other negative growth regulators, an activated p53 checkpoint may mediate the survival of cells exposed to drugs that target DNA synthesis or mitosis.

Transforming growth factor-beta3 protection of epithelial cells from cycle-selective chemotherapy in vitro.

The transforming growth factor-beta (TGF-beta) family of regulatory growth factors can reversibly arrest cell division in the G1 phase of the cell cycle. Previously, TGF-beta3 was shown to protect epithelial cells and hematopoietic cells from cytotoxic damage in vitro and in vivo, and to reduce the severity and duration of oral mucositis induced by 5-fluorouracil (5-FU) in vivo. In the present study, we tested whether TGF-beta3 can protect epithelial cells from a range of chemotherapy drugs with differing mechanisms of action, using the CCL64 cell line as a model system. We report that preincubation of cells with TGF-beta3 for 24 hr resulted in enhanced clonogenicity following exposure to vinblastine, vincristine, etoposide, taxol, ara-C, methotrexate, or 5-FU. Protection was measured in colony-forming assays, which demonstrated that the protected cells could re-enter the cell cycle and undergo multiple rounds of cell division. At high cytotoxic drug concentrations, absolute colony counts were increased for the cultures prearrested by TGF-beta3, as compared with the proliferating control cultures. The effects of TGF-beta3 were reduced for cisplatin and doxorubicin, drugs that are toxic to cells throughout the cell cycle. Thus, TGF-beta3 can effectively reduce the cytotoxicity of anticancer drugs that act predominantly in S or M phase of the cell cycle.

Bcl-2 confers growth and survival advantage to interleukin 7-dependent early pre-B cells which become factor independent by a multistep process in culture.

Early pre-B cells derived from mouse lymphoid bone marrow cultures were expanded on a surrogate stromal cell line composed of NIH3T3 fibroblasts engineered to secrete interleukin 7 (IL-7). Three immortal, IL-7-dependent cell lines were generated and infected with recombinant retroviruses to determine the effects of the human follicular B-cell lymphoma gene, bcl-2, on immature stages of B-cell development. Cells expressing bcl-2 grew at rates similar to those of control (vector only) cells when plated on bone marrow stromal lines, but exhibited a c. two-fold net proliferative advantage when grown in liquid medium supplemented with IL-7 alone. Bcl-2 prevented apoptosis when the infected early pre-B-cell lines were deprived of IL-7 and other growth factors provided by stromal cells. Following factor deprivation, a subset of cells expressing bcl-2 survived indefinitely. Two such cultures spontaneously gave rise to factor-independent variants which grew slowly in unsupplemented liquid culture and formed agar colonies, yet still responded positively to IL-7 and kit ligand, and negatively to gamma-interferon. Bcl-2 thus provides a survival capacity and modest growth advantage to early pre-B cells, which may recapitulate its effects in human B cells bearing t(14;18) translocations and ultimately contribute to transformation.

Direct stimulation of cells expressing receptors for macrophage colony-stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1.

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane-bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.

Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.

Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures.

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.

Lineage and stage specificity of isotype switching in humans.

The lineage and stage specificity of human isotype switch recombination was investigated by examining the IgH gene configuration in 61 hemopoietic malignancies representing different stages of B and T cell development. An unexpectedly high frequency (20%) of IgM-producing B cell leukemias and lymphomas had undergone CH gene rearrangements and deletions consistent with attempted switch recombination. These CH gene alterations were found on productive, non-productive, and 14q+ chromosomes. These data support the concept of a non-specific (common) switch recombinase activity that is often ineffective. No evidence of such switch recombination was found in 25 mu- or mu+ pre-B cell leukemias with the single exception of a mu- pre-B leukemia in which subsets of the cells were producing gamma- or alpha-H chains. The switch recombinase activity gamma- or alpha-H chains. The switch recombinase activity may be restricted to the B cell lineage, inasmuch as CH gene deletions were not observed in T lineage malignancies.

Bone marrow origin of a B-cell lymphoma.

To search for precursors of the neoplastic B cells in a patient with a nodular lymphoma, we produced a monoclonal antibody to a variable region idiotope on the lymphoma IgM heavy chain. Clonal ancestors of the lymphoma cells were identified by this marker among bone marrow pre-B cells (5% to 26%). A second antiidiotype (anti-Id) antibody specific for the complete lymphoma IgM kappa recognized 10% of B cells in bone marrow and blood and greater than 95% of B cells in lymphomatous lymph nodes, including one obtained after tumor conversion to a diffuse large cell lymphoma. Immunoglobulin gene analysis surprisingly revealed expansion of multiple clones of early B lineage cells in bone marrow, including members of the neoplastic clone. The data suggest that this lymphoma arose through a progression of transformational events beginning in bone marrow: first, creation of an oligoclonal pre-neoplastic pool of pre-B cells, subsequent conversion of a single subclone into low grade neoplastic B cells that homed to the lymph node follicles, and later progression to a more invasive form of the B-cell lymphoma.

Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination.

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.

Analysis with antiidiotype antibody of a patient with chronic lymphocytic leukemia and a large cell lymphoma (Richter's syndrome).

A murine monoclonal antibody made against an idiotypic determinant (Id) of surface IgM/IgD lambda molecules on chronic lymphocytic leukemia (CLL) cells of a 71-year-old woman was used for clonal analysis by two-color immunofluorescence. The anti-Id antibody identified IgM+/IgD+/lambda+ B cells as the predominant cell type of her CLL clone. In addition, substantial proportions of the IgG and IgA B cells and most of the IgM plasma cells in her bone marrow and blood were Id+. Six years after diagnosis, the patient died of respiratory failure due to infiltration of lungs by malignant cells. Autopsy revealed a dramatic change in the tumor cell morphology. The lungs, hilar nodes, and liver were infiltrated by a diffuse large cell lymphoma admixed with the leukemic cells. By immunohistologic staining these anaplastic lymphoma cells were IgM+/IgD-/lambda+ B cells expressing the same Id noted earlier on the CLL cells. The immunoglobulin gene rearrangement pattern on Southern blot analysis was also the same in leukemic blood cells and in the tissues involved by the lymphoma. Thus, the combination of antiidiotype and immunoglobulin gene analyses in this patient with Richter's syndrome revealed that a CLL clone, seemingly "frozen" in differentiation, was actually undergoing isotype switching, differentiation into plasma cells, and evolution into a rapidly growing and fetal lymphoma.