J. F. Mushinski

Selective Involvement Of Protein Kinase C Isozymes In Differentiation And Neoplastic Transformation

Publisher Summary This chapter concentrates on the present literature that implicates Protein kinase C (PKC) in the differentiation and neoplastic transformation, emphasizing the data that delineate specific roles for individual PKC isozymes. It deals generally with the PKC structure and function, and normal transduction of mitogenic signals. Despite the enormous literature, it is still not possible to define an unified view or an unanimous consensus on the role of each PKC isoform in differentiation and neoplastic transformation. The exact function of different isozymes becomes clear only when their physiological substrates are identified and characterized. Expression of the 11known PKC isozymes is often tissue specific and differentiation-stage specific. Several experimental systems, for example, erythroleukemia cells, provide clues that the differentiation is regulated by the relative concentrations of the two principal cPKCs, PKC-α and -β. Overexpression studies provide the most direct evidence that PKCs can behave as oncogenes. Considerable evidence implicates the PKC family in complex signal transduction processes. The contribution of each isoform may be inhibitory or stimulatory, but the operational word is complex.

Genes Expressed Selectively in Plasmacytomas: Markers of Differentiation and Transformation

We have analyzed a murine plasmacytoma minus highly differentiated B lymphoma subtractive cDNA library and identified eight genes that are expressed in most plasmacytomas but at a much lower level, or not at all, in most B lymphomas. Four of the genes are markers of the terminal differentiation of B lymphocytes into plasma cells: placental alkaline phosphatase, also expressed in pre-B lymphomas xlr-3, a new X-linked member of the xlr multi-gene family EGP314, a pan-epithelial glycoprotein with sequence features of an adhesion molecule PC315, a gene that is up-regulated by IL6, but without obvious sequence homologies. Two of the genes are not clearly related to normal B cell differentiation, appearing to be associated with malignant transformation of plasma cells: PC326 is a new member of the beta-transducin mosaic protein gene family. It is an X-linked gene, expressed at a very low level in testis, but in no other normal tissue, including LPS- or IL6-induced plasma cells. It has a high level of expression (apparently dysregulated) in most (> 85%) mineral oil induced plasmacytomas. However the likelihood that PC326 is expressed decreases as the tumor latency decreases when different retroviral agents are used to accelerate mineral oil induced plasmacytomagenesis. This suggests that PC326 expression may be a late event in a multi-step process of tumorigenesis. PC251 a new member of the hematopoietic growth factor receptor family, most homologous to IL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Myc-induced cyclin D2 genomic instability in murine B cell neoplasms.

I would like to begin by reiterating two important truisms of neoplasia. First, carcinogenesis, including B-cell neoplasia, is a multi-step phenomenon. That is, it involves a series of mutations. Second, these mutations must accumulate in a single cell, so it is critical that the susceptible cell be a stem cell or a cell with stem cell-like potential to self-replicate with the mutations as they accumulate. I will be considering the actions of the proto-oncogene, c-Myc, which may contribute to both these steps.

Disruption and Activation of the c-myb Locus by M-MuLV Insertion in Plasmacytoid Lymphosarcomas Induced by Pristane and Abelson Viruses

The c-myb proto-oncogene is the cellular homolog of the avian myeloblastosis virus oncogene (v-myb) (Roussel et al., 1979; Souza et al., 1980a; Bergman et al., 1981). The boundaries of c-myb are not yet known; however, studies have shown that the v-myb sequence is transduced from a portion of the coding region of c-myb into the avian myeloblastosis virus (AMV) (Klempnauer and Bishop 1983). AMV is a retrovirus that causes myeloblastic or monocytic leukemia in chickens and transforms myelomonocytic hematopoietic cells in culture (Moscovici 1975). The v-myb sequence is thought to be essential for the oncogenic potential of AMV (Duesberg et al., 1980; Souza 1980a; Gonda et al., 1981) and it appears that only certain target cells are responsive to the v-myb gene product (Moscovici 1975).

Rapid Induction of Plasmacytomas in Mice by Pristane and a Murine Recombinant Retrovirus Containing an Avian v-myc and a Defective raf Oncogene

Plasma cell tumors can be induced in genetically susceptible BALB/cAn mice by the intraperitoneal injection of various kinds of mineral (paraffin) oils or available components of these oils. Currently, the most widely used agent is pristane 2,6,10,14 tetramethy1pentadecane. The incidence of plasma cell tumors obtained in BALB/cAn mice varies according to the dose of pristane. The highest incidence of plasmacytomas occurs when mice are given three 0.5 ml i. p. injections of pristane, spaced 2 months apart (Potter and Wax 1983). Approximately 60% of the mice treated with this regimen develop plasmacytomas with a minimal latent period of 120 days and a mean latent period of 210–220 days. In contrast, when mice are given a single injection of 0.5 ml pristane i. p., only 25–30% develop plasmacytomas. The minimal latent period is 140 days and the mean latent period ca. 215 days.

Expression of the Murine Proto-Oncogene bcl-2 Is Stage Specific and Cell-Type Specific

The proto-oncogene bcl-2 is of particular interest in any consideration of neoplasia of B lymphocytes, because it has been shown to be involved in the t(14,18) (q32,q21) translocations that are found in virtually all of human follicular lymphomas, the most common B cell lymphoma in man (Bakhshi et al. 1985; Cleary and Sklar 1985; Tsujimoto et al. 1985, 1986) and in an acute pre-B lymphocytic leukemia cell line (Tsujimoto et al. 1984). The critical consequences of the interruption of the bcl-2 gene by this translocation appears to be a constitutive expression of bcl-2 transcripts that also contain sequences from the newly juxtaposed immunoglobulin heavy chain joining region (Ig-JH) (Graninger et al. 1987; Seto et al. 1988). The normal function of the bcl-2 proto-oncogene protein product is unknown, but like many other proto-oncogenes it appears to be involved in control of normal growth and development of human hemopoietic cells. Support for this comes from data that show rapid induction of bcl-2 expression in mitogen-stimulated proliferation of normal human B and T lymphocytes (Graninger et al., 1987; Reed et al., 1987; Seto et al. 1988) and a down regulation of bcl-2 expression accompanying terminal B cell maturation (Graninger et al, 1987).

Plasmacytoma Induction by J Series of v-myc Recombinant Retroviruses: Evidence for the Requirement of Two (raf and myc) Oncogenes for Transformation

In genetically susceptible BALB/cAn mice intraperitoneal injection of pristane induces plasmacytomas. More than 95% of these plasmacytomas show chromosome (6; 15) or (12;15) translocations, which result in continuous expression of c-myc (Ohno et al.1984). Direct evidence for a causative role of myc in tumor induction came from experiments using the J series of recombinant retroviruses (Rapp et al. 1985, Potter et al. 1987). The J2 and J3 viruses carry an avian MH2/MC29 hybrid v-myc and a mouse v-raf oncogene. However, in the J3 virus gag-raf gene is taken out of frame by a 256 bp deletion spanning the gag/raf border. The removal of v-raf specific sequences by this deletion leaves the minimal transforming sequence of v-raf intact (Heidecker et al., in preparation). The J5 virus carries a v-myc oncogene derived from MC29. Injection of pristane-primed mice with the J2 virus induced myeloid tumors and lymphocytic neoplasms, whereas only myeloid tumors were seen in animals inoculated with the J5 virus. Importantly, plasmacytoma development was accelerated upon infection with J3 virus.

Modes of c-myc Oncogene Activation in Murine Plasmacytomas

The murine c-myc proto-oncogene is activated and inappropriately expressed in murine plasmacytomas (PCs’) by virtue of a reciprocal 12;15 chromosomal translocation (Harris et al.,1982a; Shen-Ong et al.,1982; Crews et al.,1982; Marcu et al.,1983; Adams et al.,1983). In most PCs’, the c-myc gene is broken by the rcpt(12;15) thereby generating a complex family of aberrant myc transcripts which initiate from normally silent promoters within the first myc intron (Marcu et al.,1983; Adams et al.,1983; Stanton et al.,1983). The disruption of the c-myc locus in the PCs’ generally results in elevated levels of such truncated transcripts (Marcu et al.,1983; Mushinski et al.,1983). However, it would seem that the more general consequence of the myc associated rcpt(12;15) is the inappropriate, constitutive expression of c-myc since the normal myc allele is transcriptionally silent in these tumors (Adams et al., 1983; Stanton et al.,1983; Bernard et al.,1983).

A Retrovirus Expressing v- abl and c- myc Induces Plasmacytomas in 100% of Adult Pristane-Primed BALB/c Mice

Intraperitoneal mineral oils such as pristane induce plasmacytomas in up to 60% of inbred BALB/c mice over the course of the period of two years ( Potter et al. 1984). The appearance of the tumors can be speeded up dramatically by intraperitoneal injection of Abelson murine leukemia virus (A-MuLV) (Potter et al. 1973). However, the incidence of plasmacytomas does not increase, in part because other tumors, pre-B cell lymphomas and myeloid tumors also arise in this setting. Molecular genetic studies of plasmacytomas have revealed that 100% of these tumors show deregulated expression of the c-myc proto-oncogene (Mushinski 1988). The universal deregulation of c-myc may be necessary, but it appears to be only one step in a multistep scenario leading to plasmacytomagenesis. Recombinant retroviral vectors offer the possibility of studying the cooperation of myc with other oncogenes. So far two oncogenes, in combination with myc, have accelerated plasmacytoma induction as compared to pristane alone. These oncogene combinations, myc+raf (Troppmair et al. 1989, Kurie et al. 1990) or myc+ras (Clynes et al. 1988), in form of retroviruses, accelerated plasmacytoma onset, but did not increase the tumor incidence. Since v-abl apparently cooperated well with deregulated c-myc in A-MuLV-induced tumors in pristane-primed mice, we predicted that a retrovirus which coexpressed v-abl and c-myc would also induce plasmacytomas expeditiously. Thus the protein-encoding portion of c-myc cDNA, under the control of the widely active thymidine kinase promotor, was inserted in the genome of A-MuLV (Largaespada et al. 1990) and injected intraperitonially, either helper-free or with Moloney Murine Leukemia Virus (MoMuLV) helper virus, into immunocompetent inbred BALB/c-AnPt mice which had previously recieved a single injection of 0.5 ml pristane. When ascites accumulated and contained tumor-like cells, the mice were sacrificed and their tumors excised for transplantation, histopathological studies and for DNA and RNA extraction.

Mutations which Stabilize myc Transcripts and Enhance myc Transcription in Two Mouse Plasmacytomas

We previously reported preliminary findings concerning two mouse plasmacytomas, TEPC 1165 and TEPC 2027 which make abundant myc transcripts of 3.9 and 4.0 kb, respectively. A Northern blot of poly(A)+ RNAs from TEPC 1165 and TEPC 2027 probed with various portions of the myc gene showed that the first intervening sequence (IVS-I) ends up in mature myc mRNAs of both tumors (Mushinski 1985). All three myc gene exons are present as well.