Plasma-cell leukemia and human herpesvirus 8 infection.
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Aged↗
Biomedical subjects
S Hermouet
Publications and source records attributed to S Hermouet.
Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.
Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.
Adolescent↗
Use of pathology-specific peripheral blood CD34 thresholds to predict leukapheresis CD34 content with optimal accuracy: a bicentric analysis of 299 leukaphereses.
CD34+ cell counts in peripheral blood (PB) and corresponding numbers of CD34+ cells and colony-forming units-granulocyte/macrophage (CFU-GM) in 299 leukapheresis products of 209 patients undergoing PB progenitor cell (PBPC) mobilization for autologous transplantation in two different centers were analyzed and compared according to diagnosis: non-Hodgkin lymphoma (NHL, 94 leukaphereses), multiple myeloma (MM, 75), Hodgkin's disease (HD, 37), solid tumors (35), and chronic myeloid leukemia (CML, 32). Without separating disease entities, correlations between PB CD34+ cell counts and leukapheresis content of CD34+ cells (r>0.83, P<0.01) and CFU-GM (r>0.81, P<0.01) were excellent. In both centers, a PB CD34 threshold ensuring a leukapheresis yield > 10(6) CD34/kg was determined. This threshold was higher in center 1 than in center 2, and its predictive accuracy (91.4%, i.e., prediction correct 91.4% of the time) was significantly lower than in center 2 (98.4%, P=0.02). When data were analyzed by pathology, PB CD34+ cell counts and leukapheresis content of CD34+ cells and CFU-GM remained well correlated, and in both centers PB CD34 thresholds predictive of a yield > 10(6) CD34/kg per leukapheresis could be determined for each pathology. For most patients, pathology-specific PB CD34 thresholds could be obtained directly from the equation of the PB CD34/leukapheresis CD34 correlation curve; they varied depending on both pathology and center (range: 7-20 x 10(6) CD34/l). Pathology-specific thresholds predicted a leukapheresis yield > or = 10(6) CD34/kg accurately 100% of the time for MM patients in center 2 and HD and solid tumor patients of both centers, resulting in overall rates of accurate prediction of sufficient graft CD34 content of 96.6% in center 1 and 98.9% in center 2.
Adolescent↗
Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.
INTRODUCTION: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.
Bone Marrow Cells↗
Inheritance of chromosomally integrated viral DNA?
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Adult↗
Positive regulation of human T cell activation by Gi2 proteins and interleukin-8.
We investigated whether pertussis toxin (PT)-sensitive heterotrimeric Gi proteins (Gi1, Gi2, Gi3) are involved in the regulation of TCR-induced activation of human T cells. First, Gi proteins were inactivated by PT: pretreatment with PT of purified blood T lymphocytes before CD3 cross-linking inhibited cell proliferation (-71.1 +/- 22.0%, P < 0.001), production of interleukin-2 (IL-2; -47.3 +/- 12.6%, P = 0.008), and expression of CD25 (-24.6 +/- 11.7%, P < 0.001) and CD69 (-25.7 +/- 9.0%, P < 0.001). Then, to identify which of the three Gi was involved, Gi1, Gi2, and Gi3 proteins were specifically inactivated by stably transfecting dominant-negative mutated forms of their alpha subunit in Jurkat cells. After activation, IL-2 production and CD69 expression were inhibited only in cells expressing inactive Gi2. We then studied the effects of interleukin-8 (IL-8), a CXC-chemokine with receptors coupled to Gi2 and produced in an autocrine fashion by activated T cells. Although its effects varied among donors, exogenous IL-8 stimulated proliferation and CD25 expression (up to, respectively, 200 and 77%) of PB T lymphocytes in response to CD3 activation, in a PT-sensitive manner. IL-8 also stimulated IL-2 production (by up to 42%) and CD69 expression, although weakly (+27%). Anti-human IL-8 antibody inhibited proliferation (-43%) and CD25 up-regulation (-45%) of activated T lymphocytes. In summary, several major responses of human T lymphocytes to TCR-mediated activation are regulated by Gi2 proteins, which for this function can be activated by IL-8 in an autocrine manner.
Animals↗
Interleukin-8 and other agonists of Gi2 proteins: autocrine paracrine growth factors for human hematopoietic progenitors acting in synergy with colony stimulating factors.
We have reviewed the current knowledge on CXC chemokine interleukin-8 (IL-8) and human hematopoiesis, and more generally on agonists of heterotrimeric Gi2 proteins as regulators of human hematopoiesis. It appears that low doses of IL-8, a Gi2-agonist produced in an autocrine fashion by normal hematopoietic progenitors, mature blood cells and leukemic cells, promotes cell survival or/and proliferation in response to hematopoietic cytokines. More importantly, inactivation of the IL-8/Gi2 pathways inhibits CD34+ cell proliferation and colony formation. Similar positive effects on hematopoiesis of other, physiological or pathological, agonists of Gi2 proteins are discussed, as well as the molecular pathways involved and the consequences of activation of other G proteins (Gq, G16) by IL-8 and other Gi2-agonists.
Autocrine Communication↗
Regulation by Gi2 proteins of v-fms-induced proliferation and transformation via Src-kinase and STAT3.
We previously showed that Gi2 proteins interfere with the transduction of CSF-1 receptor (CSF-1R) proliferation signals (Corre and Hermouet, 1995). To identify CSF-1R pathways controlled by Gi2, we transfected v-fms, the oncogenic equivalent of CSF-1R, in NIH3T3 cells in which Gi2 proteins were inactivated by stably expressing a dominant negative mutant form of the alpha subunit of Gi2 (alpha i2-G204A). Expression of alpha i2-G204A resulted in decreased Src-kinase activity, delayed activation of p42 ERK-MAPK, decreased cyclin D1 expression and reduced proliferation in response to serum. In alpha i2-G204A cells transfected with v-fms, Src-kinase activity remained deficient but p42 MAPK activity and cyclin D1 expression were similar to those of vector/v-fms cells, suggesting that v-fms bypasses Src to activate the ERK-MAPK cascade. However, DNA synthesis and focus formation were inhibited by up to 80% in alpha i2-G204A/v-fms cells compared to vector/v-fms cells. We found that tyrosine phosphorylation of STAT3, also activated by CSF-1R/v-fms, was inhibited in alpha i2-G204A/v-fms cells; in addition, expression of an 85 kDa, C-terminal truncated form of STAT3 (STAT3 delta) was constitutively increased. Both the inhibition of v-fms-induced STAT3 tyrosine phosphorylation and the increased expression of STAT3 delta were reproduced by transfecting a dominant negative mutant of Src. Last, we show that expression of STAT3 delta 55C, a mutant form of STAT3 lacking the last 55 C-terminal amino acids, is sufficient to inhibit DNA synthesis and v-fms-induced transformation in NIH3T3 cells. In summary, adequate regulation by Gi2 proteins of the activity of both Src-kinase and STAT3 is required for optimal cell proliferation in response to CSF-1R/v-fms.
3T3 Cells↗
Interleukin-8: an autocrine/paracrine growth factor for human hematopoietic progenitors acting in synergy with colony stimulating factor-1 to promote monocyte-macrophage growth and differentiation.
We previously reported that alteration of the function of heterotrimeric Gi2 proteins altered proliferation of murine macrophages in response to colony stimulating factor-1 (CSF-1). Here we show that a Gi2 agonist, C-X-C chemokine interleukin-8 (IL-8), regulates monocyte-macrophage growth and differentiation. In the absence of serum, IL-8 (10 ng/mL) synergized with CSF-1 to stimulate murine monocyte-macrophage proliferation, enhanced proliferation of purified human CD34+ cells and increased the number and size of CSF-1-induced monocyte-macrophage colonies formed by purified CD34+ cells in semisolid medium. Next, as both CD34+ cells and monocyte-macrophages can produce IL-8, we used an anti-human IL-8 antibody to block an eventual activation of IL-8 receptors by autocrine IL-8. Preincubation with anti-human IL-8 antibody (20-40 microg/mL) inhibited the proliferation as well as the monocyte-macrophage colony clonogenicity of purified human CD34+ cells. Hence, in addition to being a powerful neutrophil chemoattractant, IL-8 also acts as an autocrine/paracrine growth factor for human hematopoietic progenitors, promoting the growth and differentiation of cells of monocytic lineage.
Animals↗
Reproducible scoring of CFU-GM and BFU-E grown in collagen-based semisolid medium after a short (3 h) training.
Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.
Analysis of Variance↗
Endogenous erythroid and megakaryocytic colony formation in serum-free, cytokine-free collagen gels.
We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV, 76 patients) or essential thrombocythemia (ET, 27 patients) were grown in collagen-based, serum-free, cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific, as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV, with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with > or =2 minor or 1 major PV criterion. Similarly, endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET, with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with > or =2 ET criteria. In addition, we found that in collagen gels, tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET, as these tests were positive for, respectively, 21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary, serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays.
Bone Marrow↗
Use of collagen for standardization of PBSC graft quality evaluation: a multicenter comparative analysis of commercial collagen-based and methylcellulose-based colony-forming unit (CFU) assay kits.
The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an essential test in evaluation of the quality of autologous grafts of hematopoietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available culture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio1d) and one was collagen-based (EasyClone-Multi). Using fresh and frozen samples of PBSC grafts, we compared CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using the EasyClone kit to each of the methylcellulose kits. BFU-E and CFU-GM clonogenicity of both fresh and frozen PBSC was clearly inferior with the H4431 kit, which provides conditioned medium only. CFU-GM numbers obtained with fresh and frozen PBSC samples were significantly higher with the EasyClone kit than with the H4434 and StemBio kits. BFU-E numbers were also higher with the EasyClone kit, but only when colonies were scored after May-Grünwald-Giemsa (MGG) staining. Finally, although the H4435 kit provides higher doses of recombinant cytokines than the EasyClone kit, CFU-GM and BFU-E numbers obtained for fresh or frozen PBSC with both kits were similar. In addition, CFU-GM and BFU-E numbers correlated well with CD34+ cell numbers for all five kits for both fresh and frozen PBSC. In summary, our study shows that the EasyClone-Multi and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining and storage, which facilitates colony identification and, more importantly, makes gel exchange possible for standardization of the CFU-GM assay.
Adolescent↗
G alpha16 protein expression is up- and down-regulated following T-cell activation: disruption of this regulation impairs activation-induced cell responses.
The role of heterotrimeric G proteins in T-cell activation is poorly understood. Here we show that in normal, mature human T-cells, expression of G alpha16, the 43 kDa alpha subunit of G16, varies widely, depending on T-cell activation status. Quiescent blood lymphocytes strongly up-regulate G alpha16 after Leuco A stimulation: protein expression of G alpha16 is maximal at day 4, then decreases. Consistently, in human T-cell clones, expression of G alpha16 is high in the first week following activation and decreases rapidly within the second week. In addition, permanent disruption of regulated G alpha16 expression in Jurkat T-cells by stable overexpression of 43 kDa G alpha16 inhibited Leuco A-induced interleukin-2 production, CD69 up-regulation and cell apoptosis (by 58%, 46% and 74%, respectively), suggesting that coordinate regulation of G alpha16 expression is necessary for optimal activation-induced T-cell responses, and that G alpha16 proteins may be involved in the negative regulation of TCR signalling.
Antigens, CD↗
Effect of activating and inactivating mutations of Gs- and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes.
Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gs alpha vs. Gi2 alpha, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gs alpha or the inactivating G226A(H21a)-Gs alpha point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2 alpha wild type or the missense mutations R179E-Gi2 alpha, Q205L-Gi2 alpha, and G204A(H21a)-Gi2 alpha. The activating [R201C]Gs alpha-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-L1 adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gs alpha-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2 alpha slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2 alpha and [Q205L]Gi2 alpha over differentiated controls. The inactivating [H21a]Gi2 alpha-mutant obliterated all signs of preadipocyte differentiation. It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process.
3T3 Cells↗
Analysis of platelet recovery after autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products.
We studied platelet recovery in relation to graft content in CFUs and CD34+ cells in 31 patients with multiple myeloma (21) or non-Hodgkin lymphoma (10) receiving marrow-ablative therapy followed by autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products. Twelve patients had prolonged post-transplantation thrombopenia (> or = 14 days): their graft contents in CD34+ cells, CFU-GM and BFU-E were significantly inferior to those of patients with rapid platelet recovery. Although numbers of infused CD34+ cells and CFU-GM or BFU-E were well correlated, the graft content in CD34+ cells was the only parameter predictive of platelet recovery (r = -0.38, p = 0.04), with a threshold of 2.5 x 10(6) CD34+ cells/kg. However, because rapid platelet reconstitution was obtained for 4 of 16 patients re-infused with < 2.5 x 10(6) CD34+ cells/kg, we investigated whether the graft CFU-MK content might be a better predictor of platelet reconstitution than the CD34+ cell content. Eighteen CD34 grafts were studied for CFU-MK content: CD34 and CFU-MK contents were weakly correlated (r = 0.52, p = 0.03), but there was no correlation between numbers of infused CFU-MK and time to platelet recovery. We conclude that, for autologous CD34 grafts, CFU-MK assays, like CFU-GM or BFU-E assays, cannot be used to predict platelet recovery. A CD34+ cell content > or = 2.5 x 10(6)/kg remains the only reliable indicator of the platelet reconstitution capacity of a CD34 graft.
Adult↗
[At the crossroads of memory: music].
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Aged↗
Specific expression of heterotrimeric G proteins G12 and G16 during human myeloid differentiation.
To evaluate expression of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) in human myeloid cells, we studied expression at the protein level of their alpha subunits (G alpha, the subunits responsible for the name and specificity of G proteins) in normal human myeloid progenitors and mature blood cells. We found that G alpha(s), G alpha(i2), and G alpha(q/11) proteins were expressed at high levels at all stages of granulomonocytic and erythroid differentiation, whereas expression of G alpha12 and G alpha16 proteins in normal myeloid cells was lineage-specific. G alpha12 proteins were expressed in erythroid progenitors, monocytes, and platelets, but not in normal granulocytic cells. This lineage specificity was lost in leukemic cells: G alpha12 proteins were found in human leukemic cells of both granulocytic and erythroid lineages. G alpha16 proteins were revealed in myeloid cells as two bands (43 and 46 kD), implying that G alpha16 exist in short and long forms. The 43-kD form was predominant in normal granulomonocytic cells, whereas erythroid progenitors and platelets expressed mostly the 46-kD form. Both forms of G alpha16 proteins varied during cell differentiation: in normal hematopoietic cells, G alpha16 protein expression was high in CD34+ cells, then decreased sharply during granulocytic and erythroid differentiation. In leukemic granulocytic HL60 and NB4 cells, downregulation of G alpha16 proteins was an early event (8 hours) in the process of neutrophil differentiation; in contrast, expression of G alpha16 proteins remained high during normal monocytic differentiation and in HL60 cells differentiating into monocytes with phorbol myristate acetate (PMA) or gamma-interferon (IFNgamma). Finally, we found that primary myeloid leukemia blasts, as well as leukemic cell lines, expressed G alpha16 proteins at levels higher than those found in normal CD34+ progenitors. These observations suggest that it would be worthwhile to investigate a possible role for G alpha12 and G alpha16 proteins in the regulation of human myelopoiesis.
Amino Acid Sequence↗
In vitro and in vivo growth inhibition of murine melanoma K-1735 cell by a dominant negative mutant alpha subunit of the Gi2 protein.
In murine and rat fibroblasts, activation of several G proteins (Gi2, Gq, G12) can stimulate cell growth or transformation, and can induce tumor formation in nude mice; contrastingly, inactivation of Gi2 inhibits fibroblast proliferation in vitro. We investigated whether it is possible to modulate malignant cell growth in vitro and in vivo through alteration of Gi2 protein function. To do so, we introduced mutated alpha subunits of Gi2 (alpha i2) in CL19 cells, a clone of the murine melanoma cell line K-1735. When we did this, a constitutively activated mutant (alpha i2-Q205L) and a dominant negative mutant (alpha i2-G204A) of alpha i2 were stably expressed in CL19 cells. We found that the in vitro motility of all alpha i2-transfected CL19 cells was increased; however, overexpression and alteration of the function of Gi2 did not increase metastasis formation by CL19 cells in nude mice. Expression of alpha i2-Q205L conferred a limited growth advantage to CL19 cells in vitro; in vivo, tumor formation and size, and overall survival of animals injected with CL19 cells expressing alpha i2-Q205L, were similar to controls. In contrast, expression of the inactive alpha i2-G204A mutant inhibited CL19 growth in vitro by at least 50% in all conditions tested, and mice injected with cells expressing the alpha i2-G204A mutant showed delayed tumor formation, reduced tumor size, and longer survival. We conclude that Gi2 proteins contribute to malignant cell growth, and more importantly, that inactivation of Gi2 proteins can inhibit proliferation of melanoma cells and possibly of other malignant cells both in vitro and in vivo.
Animals↗