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Follicular dendritic cells (FDCs), an essential component of the lymph node microenvironment, regulate and support B-lymphocyte differentiation, survival, and lymphoma progression. Here, we demonstrate that adhesion of mantle cell lymphoma and other non-Hodgkin lymphoma cells to FDCs reduces cell apoptosis and is associated with decreased levels of the proapoptotic protein, Bim. Bim down-regulation is posttranscriptionally regulated via up-regulation of microRNA-181a (miR-181a). miR-181a overexpression decreases, whereas miR-181a inhibition increases Bim levels by directly targeting Bim. Furthermore, we found that cell adhesion–up-regulated miR-181a contributes to FDC-mediated cell survival through Bim down-regulation, implicating miR-181a as an upstream effector of the Bim-apoptosis signaling pathway. miR-181a inhibition and Bim upregulation significantly suppressed FDC-mediated protection against apoptosis in lymphoma cell lines and primary lymphoma cells. Thus, FDCs protect B-cell lymphoma cells against apoptosis, in part through activation of a miR-181a–dependent mechanism involving down-regulation of Bim expression. We demonstrate, for the first time, that cell-cell contact controls tumor cell survival and apoptosis via microRNA in mantle cell and other non-Hodgkin lymphomas. Regulation of microRNAs by B-cell–FDC interaction may support B-cell survival, representing a novel molecular mechanism for cell adhesion–mediated drug resistance and a potential therapeutic target in B-cell lymphomas.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas. Although several protein-coding genes are altered, expression signature and importance of microRNA (miRNA) have not been well documented in this malignancy. Here, we performed miRNA expression profile in 30 patients with MCL using a platform containing 515 human miRNAs. Eighteen miRNAs were down-regulated and 21 were up-regulated in MCL compared with normal B lymphocytes. The most frequently altered miRNAs are decrease of miR-29a/b/c, miR-142-3p/5p, and miR-150 and increase of miR-124a and miR-155. Notably, expression levels of miR-29 family are associated with prognosis. The patients with significant down-regulated miR-29 had short survival compared with those who express relatively high levels of miR-29. The prognostic value of miR-29 is comparable with the Mantle Cell Lymphoma International Prognostic Index. Furthermore, we demonstrate miR-29 inhibition of CDK6 protein and mRNA levels by direct binding to 3′-untranslated region. Inverse correlation between miR-29 and CDK6 was observed in MCL. Because cyclin D1 overexpression is a primary event and exerts its function through activation of CDK4/CDK6, our results in primary MCL cells indicate that down-regulation of miR-29 could cooperate with cyclin D1 in MCL pathogenesis. Thus, our findings provide not only miRNA expression signature but also a novel prognostic marker and pathogenetic factor for this malignancy.

Drug resistance remains a critical problem in the treatment of patients with multiple myeloma. Recent studies have de-termined that Notch signaling plays a major role in bone marrow (BM) stroma-mediated protection of myeloma cells from de novo drug-induced apoptosis. Here, we investigated whether pharmacologic inhibition of Notch signaling could affect the viability of myeloma cells and their sensitivity to chemotherapy. Treatment with a γ-secretase inhibitor (GSI) alone induced apoptosis of myeloma cells via specific inhibition of Notch signaling. At concentrations toxic for myeloma cell lines and primary myeloma cells, GSI did not affect normal BM or peripheral blood mononuclear cells. Treatment with GSI prevented BM stroma-mediated protection of myeloma cells from drug-induced apoptosis. The cytotoxic effect of GSI was mediated via Hes-1 and up-regulation of the proapoptotic protein Noxa. In vivo experiments using xenograft and SCID-hu models of multiple myeloma demonstrated substantial antitumor effect of GSI. In addition, GSI significantly improved the cytotoxicity of the chemotherapeutic drugs doxorubicin and melphalan. Thus, this study demonstrates that inhibition of Notch signaling prevents BM-mediated drug resistance and sensitizes myeloma cells to chemotherapy. This may represent a promising approach for therapeutic intervention in multiple myeloma.

Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1arrest associated with elevated p27Kip1and p21 (WAF1) proteins. The adhesion-mediated p27Kip1and p21increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

Preliminary studies have shown valspodar (PSC‐833: Novartis Pharmaceuticals, East Hanover, NJ) to be a potent inhibitor of multidrug resistance (MDR), one cause of resistance to chemotherapy. An international randomized control study (Phase III) evaluated the use of vincristine, doxorubicin, and dexamethasone (VAD) with (n = 46) and without (n = 48) valspodar in the treatment of patients with recurring or refractory multiple myeloma.Patients with documented recurrence or refractory myeloma were stratified based on prior treatment exposure and creatinine and randomized. Because of interaction of valspodar with vincristine and doxorubicin, the doses of these drugs were reduced compared with the VAD‐alone arm, and the doxorubicin was further reduced in the last 15 patients when given with valspodar based on pharmacokinetic and toxicity studies.There were no complete or near‐complete responses. There were 29% partial responses (PRs) in the VAD‐alone arm and 44% with valspodar (P = 0.2). Median progression‐free survival was 7 months with VAD alone and 4.9 months with valspodar (P = 0.50). Subjective response was 19% with VAD alone and 17% with valspodar (P = 1.0). Median survival with VAD alone was 18.5 months and 15.3 with the addition of valspodar (P = 0.055). Toxicity of Grade 3 or greater was higher (P < 0.0001) in the valspodar arm (89%) compared with the VAD‐alone arm (58%). The reduction of doxorubicin dose reduced toxicity but not significantly (P = 0.11).The addition of the MDR‐modulating agent valspodar to VAD did not improve treatment outcome. Toxicity was increased in the valspodar‐treated group compared with VAD alone. Cancer 2006. © 2006 American Cancer Society.

Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). In this report, we demonstrate that enhanced interstrand cross-link (ICL) repair via the Fanconi anemia (FA)/BRCA pathway contributes to acquired drug resistance in melphalan-resistant myeloma cell lines, and disruption of this pathway reverses drug resistance. Using the alkaline comet assay (single-cell gel electrophoresis), we observed that melphalan-resistant cells have reduced ICL formation and enhanced ICL repair compared with melphalan-sensitive cells. Cell-cycle studies demonstrated that enhanced ICL repair released cells from melphalan-induced cell-cycle delay. Using siRNA to knock down FANCF in 8226/LR5 and U266/LR6 drug-resistant cells demonstrated a direct relationship between ICL repair capacity and drug sensitivity. Overexpression of FANCF in 8226/S and U266/S drug-sensitive cells partially reproduced the drug-resistant phenotype. These data show that enhanced DNA repair via the Fanconi anemia/BRCA pathway is involved in acquired melphalan resistance. Our findings provide for a new target to enhance response to DNA cross-linking agents in cancer treatment. (Blood. 2005;106:698-705)

The bone marrow (BM) microenvironment plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of Notch family proteins in myeloma and other malignant lymphoid cell line growth and response to chemotherapeutic drugs. All 8 tested cell lines expressed Notch-3 and Notch-4; 7 cell lines expressed Notch-1; and 6 expressed Notch-2 proteins. Interaction with BM stroma (BMS) activated Notch signaling in tumor cells. However, activation of only Notch-1, but not Notch-2, resulted in protection of tumor cells from melphalan- and mitoxantrone-induced apoptosis. This protection was associated with up-regulation of p21WAF/Cip and growth inhibition of cells. Overexpression of Notch-1 in Notch-1- U266 myeloma cells up-regulated p21 and resulted in protection from drug-induced apoptosis. Thus, this is a first report demonstrating that Notch-1 signaling may be a primary mechanism mediating the BMS influence on hematologic malignant cell growth and survival. (Blood. 2004; 103:3503-3510)

Patients with multiple myeloma (MM) with mutated RAS are less likely to respond to chemotherapy and have a shortened survival. Therefore, targeting RAS farnesylation may be a novel approach to treatment of MM. We evaluated the activity and tolerability of the farnesyltransferase (FTase) inhibitor tipifarnib (Zarnestra) in a phase 2 trial as well as its ability to inhibit protein farnesylation and oncogenic pathways in patients with relapsed MM. Forty-three patients (median age, 62 years [range, 33-82 years]) with a median of 4 (range, 1-6) chemotherapy regimens entered the study. Tipifarnib, 300 mg orally twice daily, was administered for 3 weeks every 4 weeks. The most common toxicity was fatigue occurring in 66% of patients. Other toxicities included diarrhea, nausea, neuropathy, anemia, and thrombocytopenia. Sixty-four percent of the patients had disease stabilization. Treatment with tipifarnib suppressed FTase (but not geranylgeranyltransferase I) in bone marrow and peripheral blood mononuclear cells and also inhibited the farnesylation of HDJ-2 in unfractionated mononuclear cells and purified myeloma cells. Inhibition of farnesylation did not correlate with disease stabilization. Finally, tipifarnib decreased the levels of phosphorylated Akt and STAT3 (signal transducer and activator of transcription 3) but not Erk1/2 (extracellular signal regulated kinase 1 and 2) in bone marrow cells. We conclude that tipifarnib is tolerable, can induce disease stabilization, and can inhibit farnesylation and oncogenic/tumor survival pathways. (Blood. 2004;103:3271-3277)

We previously showed that adhesion of myeloma cells to fibronectin (FN) by means of β1 integrins causes resistance to certain cytotoxic drugs. The study described here found that adhesion of U937 human histiocytic lymphoma cells to FN provides a survival advantage with respect to damage induced by the topoisomerase (topo) II inhibitors mitoxantrone, doxorubicin, and etoposide. Apoptosis induced by a topo II inhibitor is thought to be initiated by DNA damage. The neutral comet assay was used to determine whether initial drug-induced DNA damage correlated with cellular-adhesion–mediated drug resistance. Cellular adhesion by means of β1 integrins resulted in a 40% to 60% reduction in mitoxantrone- and etoposide-induced DNA double-strand breaks. When the mechanisms regulating the initial drug-induced DNA damage were examined, a β1 integrin–mediated reduction in drug-induced DNA double-strand breaks was found to correlate with reduced topo II activity and decreased salt-extractable nuclear topo IIβ protein levels. Confocal studies showed changes in the nuclear localization of topo IIβ; however, alterations in the nuclear-to-cytoplasmic ratio of topo IIβ in FN-adhered cells were not significantly different. Furthermore, after a high level of salt extraction of nuclear proteins, higher levels of topo IIβ–associated DNA binding were observed in FN-adhered cells than in cells in suspension. Together, these data suggest that topo IIβ is more tightly bound to the nucleus of FN-adhered cells. Thus, FN adhesion by means of β1 integrins appears to protect U937 cells from initial drug-induced DNA damage by reducing topo II activity secondarily to alterations in the nuclear distribution of topo IIβ.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.

Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (?4ß1) and VLA-5 (?5ß1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in ?4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and ?4-low phenotype. Whether VLA-4–mediated FN adhesion offers a survival advantage over VLA-5–mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.

We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95-mediated apoptosis (Landowski et al: Blood89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and drug-induced apoptosis occurs only when cytotoxic drugs are used as the selecting agent. Using the inverse approach, we transfected the CD95-negative cell line, K562, with a CD95 expression vector. Clones expressing variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95 surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show that CD95 and chemotherapeutic drugs use common apoptotic effectors, but the point of convergence in these two pathways is downstream of CD95 receptor/ligand interaction.

Classically, studies of drug resistance in cancer have focused on the molecular biology of single cancer cells. These types of studies have provided important information regarding certain drug resistance mechanisms, including mechanisms that reduce intracellular drug accumulation, alter or repair drug-induced damage, and reduce drug-induced apoptosis. While these cellular mechanisms undoubtedly contribute to the overall phenomenon of drug resistance, it is now evident that the tumor cell microenvironment also influences how a tumor cell behaves and responds to cytotoxic drugs or radiation. Two different forms of tumor cell-environmental interaction may explain how some tumor cells survive initial drug exposure and eventually express classical mechanisms of drug resistance. The first form involves soluble mediators, such as interleukins, that are secreted by non-tumor, stromal cells. Interleukin-6 (IL-6) is a classical example of how a soluble mediator secreted by the tumor microenvironment is capable of enhancing tumor cell survival and perhaps blocking apoptosis. The second form of tumor cell-environment interaction requires direct cell contact and has been given the term cell-adhesion-mediated drug resistance (CAM-DR). In this case, binding extracellular matrix ligands in the tumor microenvironment may activate cell adhesion molecules, such as the integrins, and these interactions result in the activation of signal transduction pathways that block drug-induced apoptosis. Interrupting the tumor cell-environment interactions or the associated signal transduction pathways may represent a new approach for the treatment of cancer. Copyright 1999 Harcourt Publishers Ltd

CD95 (Fas)-induced apoptosis plays a critical role in the elimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS) and lpror gldmice and attributed to CD95 and CD95L gene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation of antigen-activated cytotoxic T lymphocytes. Autoimmune features such as hypergammaglobulinemia, rheumatoid factor, and circulating immune complexes are common features in LGL leukemia and ALPS. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to a defective CD95 apoptotic pathway. In this study, we investigated expression of CD95 and CD95L in 11 patients with CD3+LGL leukemia and explored the apoptotic response to agonistic CD95 monoclonal antibody (MoAb). We found that leukemic LGL from each patient expressed constitutively high levels of CD95/CD95L, similar to those seen in normal activated T cells. However, cells from 9 of these 11 patients were totally resistant to anti-CD95–induced apoptosis. Similarly, cells were resistant to anti-CD3-MoAb–triggered cell death. Lack of anti-CD95–induced apoptosis was not due to mutations in the CD95 antigen. Leukemic LGL were not intrinsically resistant to CD95-dependent death, because LGL from all but 1 patient underwent apoptosis after phytohemagglutinin/interleukin-2 activation. The patient whose leukemic LGL were intrinsically resistant to CD95 had an aggressive form of LGL leukemia that was resistant to combination chemotherapy. These findings that leukemic LGL are resistant to CD95-dependent apoptosis despite expressing high levels of CD95 are similar to observations made in CD95L transgenic mice. These data suggest that LGL leukemia may be a useful model of dysregulated apoptosis causing human malignancy and autoimmune disease.

Verapamil was evaluated as a chemosensitizer for reversing multidrug resistance in multiple myeloma both in vitro and in clinical trials. Bone marrows from 59 myeloma patients in relapse were evaluated for several resistance parameters: expression of p-glycoprotein (MDR1), doxorubicin (Adriamy-cin) and vincristine sensitivity, and the ability of added verapamil to reduce resistance to the cytotoxic agents. We found that verapamil was capable of sensitizing myeloma cells that exhibited resistance to doxorubicin and vincristine in vitro, but did not enhance sensitivity of cells that were drug sensitive (P≪ .001). Myeloma cells expressing MDR1 immunohistochemically tended to be more doxorubicin resistant in vitro than MDR1-negative cells. In the clinical trials, 22 patients with myeloma refractory to vincristine-Adriamycin-dexamethasone (VAD) were treated with VAD plus high-dose intravenous verapamil (Ve). Among the 22 patients treated with VAD/Ve, five achieved a partial remission (23%). The median relapse-free survival for the VAD/Ve responders was 5.4 months and their overall survival from the start of VAD/Ve was better than that of the nonresponders. Among the subset of 10 patients whose myeloma cells were MDR1 positive, four responded clinically (40%), whereas none of five patients with MDR1-negative myeloma cells achieved remission with VAD/Ve. We also observed that myeloma cells from three of four VAD/Ve clinical responders exhibited in vitro chemosensitization with verapamil, whereas in vitro verapamil chemosensitization was seen in only one of six clinical nonresponders. Our observations demonstrate that clinical reversal of multidrug resistance can be achieved in some patients with VAD-refractory myeloma with the use of verapamil. In addition to their value in drug development, in vitro tests of MDR1 expression and of chemosensitizers plus cytotoxic drugs on the patients' bone marrow myeloma cells may identify patients who will respond clinically to chemosen-sitizer-containing regimens. We anticipate that chemosensitizer regimens capable of inhibiting multidrug resistance will play an increasing role in the treatment of hematologic malignancies, including B-cell neoplasms such as multiple myeloma and the non-Hodgkin's lymphomas.

Although a number of investigators have established that mitoxantrone (Novantrone®; dihydroxyanthracenedione) inhibits RNA and DNA synthesis and intercalates with DNA in vitro, its exact mechanism of action is unclear. Mitoxantrone is structurally related to a series of substituted anthraquinones and has features known to be essential for DNA intercalation; however, we have determined recently that mitoxantrone binds DNA in intact L1210 leukemia cells by a non-intercalative, electrostatic interaction and induces both protein associated and non-protein associated DNA strand scissions. The difference between mitoxantrone and doxorubicin with respect to their interactions with DNA could account for their relative lack of cross-resistance in the treatment of lymphoma and acute leukemia.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

Multidrug-resistant (MDR) myeloma patients failing chemotherapy may express P-glycoprotein (PGP), which serves as an efflux pump protecting the neoplastic cells. Unknown is whether PGP expression might relate to prior cytotoxic drug exposure. To address this question, we studied 106 consecutive bone marrow samples from 104 myeloma patients with samples studied either before or after therapy and at the time of relapse. We performed an established immunocytochemical assay of PGP using an MDR-1–specific monoclonal antibody and correlated PGP status with prior chemotherapy dosage. Myeloma patients with no prior therapy had a low incidence of PGP expression (6%, 3/47), whereas those receiving chemotherapy had a significantly higher incidence (43%, 21/49) (P <.0001). A substantially higher incidence of PGP expression (50%, 83%, respectively) occurred when the total vincristine dose exceeded 20 mg and when doxorubicin exceeded 340 mg. In the 11 patients who received both high vincristine and doxorubicin dosages (>20 mg, >340 mg total dose) there was 100% incidence of PGP expression in the tumor cells. These data provided the basis for a predictive mathematical model from which dose-related PGP expression normograms were generated. Time with myeloma for PGP-negative patients (mean 33 months) had overlapping confidence limits with PGP-positive patients (mean 42 months), suggesting that disease duration was not a significant variable. PGP expression did not correlate with other clinical factors or immunophenotypic factors. Our findings indicate a strong correlation between PGP expression in myeloma and past chemotherapy in myeloma, in particular, related to prior exposure to the natural product agents vincristine and doxorubicin. Additionally, the proportion of PGP-positive plasma cells was significantly higher in the doxorubicin-treated patients than the nondoxorubicin-treated patients (87.7% v65.17%; P= .013). Combined high vincristine and doxorubicin total dosage appear highly predictive of PGP expression.

One major obstacle to the successful treatment of epithelial derived tumors, such as breast and prostate carcinoma, is the presence of a multiple drug resistance phenotype. The drug resistance which is observed in growing epithelial derived cancer cells could either be an intrinsic, selected and/or an acquired characteristic. A survey of the survival data from several laboratories suggests that epithelial derived tumor cells, which have never been challenged with damaging agents, are in some cases 10 to 2,000 times more resistant to various chemotherapeutic agents as compared to hematopoietic cell lines. An intrinsic characteristic of epithelial cells is their resistance to the lethal effects of multiple types of damaging agents. A major feature of epithelial derived tumors is the expression of the intermediate filament type proteins known as cytokeratin. The simplest cytokeratin combination, cytokeratin 8 and 18, is a major cytoplasmic element within the cells of epithelial derived tumors. Earlier work showed that cytokeratin could be modified by mitoxantrone, a chemotherapeutic agent used in the treatment of breast cancer. Increasing data indicates that the intrinsic drug resistance phenotype is due in part to the presence of continued expression of the cytokeratin 8 and 18. The cytokeratin dependent drug resistance (C-MDR) has been observed in two different cell types that were engineered to contain cytokeratin 8 and 18 expression. The cytokeratin monomers are known to self assemble into intermediate filament networks as shown by numerous basic studies. Experiments using transfected cell lines which are unable to assemble networks indicated that C-MDR does not depend upon the formation of an intermediate filament network. Selection of cytokeratin network defective tumor cells did not increase their sensitivity to chemotherapeutic agents. These data are interesting since it suggests that the C-MDR phenotype is not dependent upon the structural nature (i.e. network forming ability) of the cytokeratin. Our current working hypothesis is that the interaction of the damaging agent with cytokeratin may initiate signaling response(s) for cell survival.