Leukemia Research Vol. 17, No. 3, pp. 291-293, 1993. 0145-2126/93 $6.00 + .00 Printed in Great Britain. © 1993 Pergamon Press Ltd

CONCISE REPORT

METHYLATION STATUS WITHIN EXON 3 OF THE c-myc GENE AS A PROGNOSTIC M A R K E R IN MYELOMA AND L E U K A E M I A

JANET STEPHENSON, RECEP AKDAG, NAMIK OZBEK and GHULAM J. MUFTI

Department of Haematology, Kings College Hospital School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, U.K.

(Received 1 November 1992. Revision accepted 7 November 1992)

Abstract--The third exon of the c-myc gene contains a CpG site which has been implicated as a regulatory region. When this site is methylated it has protein binding properties and binds a different set of proteins in normal and neoplastic cells.

Recent work using myeloma cell lines indicates a correlation between hypomethylation at this site and enhanced expression of the myc protein. We investigated the methylation status of this site in 10 cases of myeloma but found that there was no change from the high degree of methylation found in normal cells. Therefore, methylation status at this site is unlikely to serve as a prognosticator in myelomatosis. However, methylation changes at this site were observed in DNA from two cases of CMML, in which hypomethylation was observed and in three AML cases, which were completely methylated at this site.

Key words: Myeloma, CMML, AML, c-myc, methylation.

I N T R O D U C T I O N

REGULATION of c-myc expression is extremely com- plex and involves multiple mechanisms at several levels of control [1]. These include the use of multiple promotors, pausing of transcription, destabilisation of the rnyc mRNA, and both positive and negative regulation at the protein level mediated by hetero- dimer formation. The c-myc protein also has the property of binding to specific methylated DNA sites [21.

Hypomethylat ion at a specific site within the third exon encoding c-myc has been shown to correlate with increased expression of myc mRNA in myeloma cell lines [3]. Recent work on purified myeloma cells [4] is suggestive of a correlation between myc m RN A levels and the clinical behaviour of the patient's disease in cases with myelomatosis. Methylation at this site has also been shown to decrease in the progression of colon cancer from dysplasia in adeno- matous polyps to adenocarcinoma and metastasis [5].

The role of the exon 3 methylated site in the regulation of myc expression is unknown, but it pos-

Abbreviations: CMML, chronic myelomonocytic leu- kaemia; AML, acute myeloid leukaemia.

Correspondence to: J. Stephenson, Dept of Haema- tology, Kings College Hospital School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, U.K.

sesses protein binding properties, which bind a dif- ferent set of proteins in normal and dysplastic colonic cells [6]. It is conceivable that myc regulates its own expression at this site.

Assessment of methylation status at this site is thus of interest i n myeloma and other haematological disorders associated with deregulated proliferation of haemopoetic progenitors. One such instance is chronic myelomonocytic leukaemia (CMML), in which increased proliferation of the cells in the granulocytic/monocytic lineages is evident and pro- gression to acute myeloid leukaemia occurs in up to 40% of cases [7]. We have investigated the methyl- ation status of myc exon 3 in D N A from peripheral blood and/or bone marrow aspirate samples in 10 myeloma patients, 5 normal patients, 3 A M L and 2 CMML patients.

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M A T E R I A L S A N D M E T H O D S

DNA was extracted from whole blood using proteinase K digestion and phenol extraction according to standard techniques. Ten micrograms of DNA were digested at 37°C with EcoRI (5 units per #g) for 3 h and then with either MspI or HpaII (10 units per #g) overnight. Eiectrophoresis and Southern transfer was carried out according to standard protocols. The probe used was a 1.6 kb genomic ClaI/ EcoR1 fragment excised from plasmid pHSR-1 by restric- tion digestion and separation of the fragments through 0.8% low melting temperature agarose. Twenty-five nano-

292

kb

3.3

2.2

1.1

1

H

2 3 4 5

M H M H M H M H

J. STEPHENSON et al.

6 7 8 9 10 11 12

M H M H M H M H M H M H M H M

FIG. 1. H designates digestion with EcoR1 and HpalI. M designates digestion with EcoRI and MsplI. 1,5 = normal;

2,3,10-12 = myeloma; 4,6,7 = AML; 8,9 = CMML.

grams of the fragment in LMT agarose was labelled by random priming using a multiprime kit according to the manufacturer's recommendations (Amersham). Incubation with Klenow polymerase was carried out at room temperature overnight. Hybridisation was carried out according to standard protocols with washing carried out to high stringency (0.1 x SSC, 0.1% SDS). Figures for methylation were obtained by scanning using UVP gel scanning software. The intensity of signals from the 1.1 and 2.2 kb bands was compared to that of the 3.3 kb band according to the formula

[1.1 + 2.2]/[1.1 + 2.2 + 3.3]

and expressed as percentage hypomethylation.

RESULTS AND DISCUSSION

Restriction digestion at the internal Hpal I /MspI site cleaves a 3.3 kb fragment into 2.2 and 1.1 kb fragments, shown in Fig. 1. Results show that all myeloma D N A samples demonstrated methylation patterns identical to normal peripheral blood DNA, regardless of peripheral blood or bone marrow source (% hypomethylation, mean = 7.5%, S.D. = 2.3, n = 5 for normal samples; mean = 5.0%, S.D. = 2.22, n = 10 for myeloma samples). This indicates that in the majority of cells analysed this site was methyl- ated, results which are contrary to those obtained in myeloma cell lines [3]. It is unlikely that this is due to low numbers of neoplastic cells, since the percentage of marrow plasma cells varied between 25 and 90, and all patients had florid disease at the time of investigation. In view of this, hypomethylation at this region is unlikely to be a useful marker in this disease.

The two CMML samples showed hypomethylation levels of 20%, S.D. = 0.05. No cleavage by HpalI within exon 3 was observed in the three AML samples

analysed (hypomethylation less than 1%). Lineage- specific methylation changes at the c-fms locus have been observed [8] in both myeloid and lymphocytic leukemias. At this locus, hypermethylation is demon- strated in the myeloid lineage of A ML cells, with hypomethylation in lymphocytes in CLL and ALL. Cell separation into lineage-specific populations prior to D N A extraction would resolve the question of lineage-specific myc methylation, and is currently under investigation.

The observation of decreased methylation in CMML samples implies a role for c-myc in the pro- liferation of monocytes in this disorder. Additionally, the observation of hypermethylation of c-myc in three cases of A ML suggests that methylation status at this site alters during progression from CMML to AML. Expansion of this study to include greater numbers of C M M L / A M L samples is required, as is correlation of methylation status with c-myc R N A levels.

R E F E R E N C E S

1. Cole M. D. (1986) The myc oncogene: its role in trans- formation and differentiation. Ann. Rev. Genet. 20, 361.

2. Prendergast G. C. & Ziff E. B. (1991) Methylation- sensitive sequence-specific DNA binding by the c-myc basic region. Science 251, 186.

3. Ohtsuki T., Nishitani K., Hatamochi A., Yawata Y. & Namba M. (1991) Analysis of methylation in the c-myc gene in five human myeloma cell lines. Br. J. Haemat. 77, 172.

4. Nobuyoshi M., Kawano M., Tanaka H., Ishikawa H., Tanabe O., Iwato K., Asaoku H., Sakai A. & Kuramoto A. (1991) Increased expression of the c-myc gene may be related to the aggressive transformation of human myeloma cells. Br. J. Haemat.77, 523.

5. Sharrard R. M., Royds J. A., Rogers S. & Shorthouse

Exon 3 of c-myc as a prognostic marker 293

A. J. (1992) Patterns of methylation of the c-myc gene in human colorectal cancer progression. Br. J. Cancer 65, 667.

6. Sharrard R. M., Royds J. A. & Shorthouse A. J. (1991) Protein DNA interactions involving a 34 bp sequence from the third exon of the c-myc oncogene. Br. J. Cancer 63, 20.

7. Worsley A. M., Oscier D. G., Stevens J., Darlow S., Figes A., Mufti G. J. & Hamblin T. J. (1988) Prognostic features of CMML: a modified Bournemouth score gives the best prediction of survival. Br. J. Haemat. 68, 17.

8. Feigner J., Kreipe H., Heidorn K., Jaquet K., Zsehunke F., Radzun H. J. & Parwaresch M. R. (1991) Increased methylation of the c-fms protooncogene in acute mye- lomonocytic leukemias. Pathobiology 59, 293.