B-Cell lymphoma induction by akv murine leukemia viruses harboring one or both copies of the tandem repeat in the U3 enhancer

Abstract

Akv is an endogenous, ecotropic murine leukemia virus (MuLV) of the AKR strain. It has served as a prototype nonpathogenic or weakly pathogenic reference virus for studies of closely related potent lymphomagenic viruses such as the T-lymphomagenic SL3-3. We here report that Akv and an Akv mutant (Akv1-99) with only one copy of the 99-bp transcriptional enhancer induce malignant lymphomas with nearly 100% incidence and mean latency periods of 12 months after injection into newborn NMRI mice. Molecular analysis of tumor DNA showed that the majority of the tumors were of the B-cell type. Sequence analysis of proviral transcriptional enhancers in DNA of B-cell lymphomas revealed conservation of the enhancer sequence, as well as a lack of sequence duplications of the Akv1-99 variant, while the repeat copy number in Akv was subject to fluctuations. In support of a B-cell specificity of the Akv enhancer, a murine plasmacytoma cell line was found to sustain three- to fivefold-higher transient transcriptional activity upon the Akv and Akv1-99 enhancers than upon the enhancer of the T-lymphomagenic SL3-3 MuLV. Thus, the overall picture is that Akv MuLV possesses a B- lymphomagenic potential and that the second copy of the 99-bp sequence seems to be of minor importance for this potential. However, in one animal the lymphomas induced by Akv1-99 were of the T-cell type. Among the 24 tumors analyzed only this one harbored a clonal proviral integration in the c-myc locus. This provirus had undergone a duplication of a 113-bp sequence of the enhancer region, partly overlapping with the 99-bp repeat of Akv, as well as a few single nucleotide alterations within and outside the repeats. Taken together with previous studies, our results suggest that T- versus B-lymphomagenic specificity of the enhancer is governed by more than one nucleotide difference and that alterations in binding sites for transcription factors of the AML1 and nuclear-factor-1 families may contribute to this specificity.

FIG. 1

Proviral structures of Akv and Akv1- 99. Akv contains a 99-bp perfect tandem repeat in the U3 enhancer region, while Akv1- 99 contains only one copy of the 99-bp sequence. Shown is the 99-bp sequence with demarcation of NF-1 sites 1 and 2 and the Akv core and E-box sequences.

FIG. 2

Tumor classification by Southern hybridizations. (A) DNA from Akv-induced tumors 2, 3, and 5 and Akv1- 99-induced tumors 3, 4, 9, and 10 was cleaved by HindIII and analyzed by using the TCRβ J1 and TCRβ J2 probes. (B) DNA from Akv-induced tumors 1, 2, 3, 4, 5, 6, and 7 and Akv1- 99-induced tumors 1, 2, 3, 4, 5, 6, 7, and 10 was cleaved by EcoRI and analyzed by using IgH probe. The significance of the tendency towards lower intensity of the IgH germ line band for the Akv1- 99 tumors than for the Akv tumors is not clear. (C) DNA from Akv1- 99-induced tumors 2, 1, 3, 4, 9, and 10 and Akv-induced tumors 8, 9, 10, 1, 4, and 6 was cleaved by HindIII and analyzed by using the Igκ probe. Arrows mark the position of the unrearranged genomic band. Size markers of 9.4, 6.5, and 2.3 kb are indicated. The location of the probes in relation to the joining regions is indicated by a horizontal black bar in the diagrams below each panel.

FIG. 2

Tumor classification by Southern hybridizations. (A) DNA from Akv-induced tumors 2, 3, and 5 and Akv1- 99-induced tumors 3, 4, 9, and 10 was cleaved by HindIII and analyzed by using the TCRβ J1 and TCRβ J2 probes. (B) DNA from Akv-induced tumors 1, 2, 3, 4, 5, 6, and 7 and Akv1- 99-induced tumors 1, 2, 3, 4, 5, 6, 7, and 10 was cleaved by EcoRI and analyzed by using IgH probe. The significance of the tendency towards lower intensity of the IgH germ line band for the Akv1- 99 tumors than for the Akv tumors is not clear. (C) DNA from Akv1- 99-induced tumors 2, 1, 3, 4, 9, and 10 and Akv-induced tumors 8, 9, 10, 1, 4, and 6 was cleaved by HindIII and analyzed by using the Igκ probe. Arrows mark the position of the unrearranged genomic band. Size markers of 9.4, 6.5, and 2.3 kb are indicated. The location of the probes in relation to the joining regions is indicated by a horizontal black bar in the diagrams below each panel.

FIG. 2

Tumor classification by Southern hybridizations. (A) DNA from Akv-induced tumors 2, 3, and 5 and Akv1- 99-induced tumors 3, 4, 9, and 10 was cleaved by HindIII and analyzed by using the TCRβ J1 and TCRβ J2 probes. (B) DNA from Akv-induced tumors 1, 2, 3, 4, 5, 6, and 7 and Akv1- 99-induced tumors 1, 2, 3, 4, 5, 6, 7, and 10 was cleaved by EcoRI and analyzed by using IgH probe. The significance of the tendency towards lower intensity of the IgH germ line band for the Akv1- 99 tumors than for the Akv tumors is not clear. (C) DNA from Akv1- 99-induced tumors 2, 1, 3, 4, 9, and 10 and Akv-induced tumors 8, 9, 10, 1, 4, and 6 was cleaved by HindIII and analyzed by using the Igκ probe. Arrows mark the position of the unrearranged genomic band. Size markers of 9.4, 6.5, and 2.3 kb are indicated. The location of the probes in relation to the joining regions is indicated by a horizontal black bar in the diagrams below each panel.

FIG. 3

Analysis of proviral U3 regions in tumor DNAs. (A) PCR amplification of U3 enhancer sequences of DNA of Akv- and Akv1- 99-induced tumors; the numbers refer to the animal numbers presented in Table 2. M, DNA size markers. The positions of amplified fragments resulting from Akv (positions 12 to 375) and Akv1- 99 (positions 12 to 276) are shown. (B) Akv and Akv1- 99 U3 structures showing the PCR primers used in panel A. Below is shown the input Akv1- 99 sequence (positions 40 to 258). (C) All nucleotide sequences of PCR fragments of Akv1- 99-induced tumors were determined from positions corresponding to 40 to 258 of the input U3 sequence shown in panel A. Only tumor 10 showed changes as shown in the lower band.

FIG. 4

Analysis of a provirus integrated at the c-myc locus in Akv1- 99 tumor 10. (A) Integration site and orientation of provirus at c-myc. Indicated are the c-myc probe used for Southern hybridization, the c-myc-specific primers a, b, and c used for PCR analysis, and the provirus-specific primers d and e. The proviral site of integration and orientation was determined by sequence analysis of PCR products with the primers described in Materials and Methods. The position numbering corresponds to a c-myc promoter sequence from GenBank and EMBL (nucleotide sequence accession no. M12345). (B) Nucleotide sequence of the complete U3 region of the c-myc integrated provirus. The nucleotide sequence was determined from PCR amplification products of provirus–c-myc junction fragments with LTR-specific primers as described in Materials and Methods. Marked above the sequence are nucleotide differences in Akv1- 99 and the location of the 99-bp tandem repeat of Akv. The arrows underlining the sequence mark an imperfect tandem repeat of 113(115) bp. Two nucleotide insertions in the second repeat are marked with (−). (C) U3 repeat organization of Akv and the c-myc integrated provirus of Akv1- 99 10. Differences in core and NF-1 sites are shown.

FIG. 5

Analysis of the presence of specific U3 sequences in DNA of virus-induced tumors and noninfected control tissues. The upper panel shows the U3 region, with the arrows indicating the repeated sequence in Akv and Akv1- 99 10. Also shown are the four primer sets used to verify the nonendogenous nature of the altered U3 region in the Akv1- 99-induced tumor 10. The lower panel shows the results of PCRs on DNA of the two Akv-induced tumors 1 and 2, the two Akv1- 99-induced tumors 9 and 10, and the tissues from two mock-injected control mice. +, Amplified fragments of the expected size were detected; −, no amplified bands were detected.