Published in: J. Virol. vol. 75, no. 20, pp. 9977-9982 (October, 2001):
DOI: 10.1128/JVI.75.20.9977-9982.2001
http://jvi.asm.org/cgi/content/abstract/75/20/9977


"Replacement of the Epstein-Barr Virus Plasmid with the EBER Plasmid in Burkitt's Lymphoma Cells".

Seiji Maruo, Asuka Nanbo, and Kenzo Takada*

Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido
University, N15 W7, Kita-ku, Sapporo 060-0815, Japan

* Corresponding author. Mailing Address: Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo 060-0815, Japan.
Phone: 81-11-706-5071.    Fax: 81-11-717-1128.
E-mail:   kentaka@med.hokudai.ac.jp


Abstract:
Introduction:
Methods and Results:
   Detection of DNA species, EBER,  and EBNA:
   EBER expression in EBER-transfected Akata cell clones:
   Detection of DNA from the EBV plasmid and transfected EBER DNA in Mutu I cell clones:
   Effect of EBER expression on retention of oriP-containing plasmid:
Conclusions:
Acknowledgments:
References:
RNA Data Base Entry:
Additional References:
Other Links:
Further Information and Feedback:

Abstract:

Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.


Epstein-Barr virus (EBV) is present in tumor cells of more than 90% of cases of Burkitt's Lymphoma (BL) in areas of endemicity in equatorial Africa and New Guinea, as well as in tumor cells of about 10% of sporadic cases of BL occurring worldwide [9]. However, the role of EBV in the genesis of BL is still not fully understood. To gain insight into this problem, we have investigated the pathogenic role of EBV in BL cell line Akata. The Akata cell line is an EBV-positive cell line derived from a Japanese patient [13]. It retains BL-type EBV expression (termed type I latency) [12], which is characterized by by expression of a restricted set of latent genes , including EBV nuclear antigen 1 (EBNA1), EBV-encoded RNAs (EBERs), (specifically EBER-1 and -2), transcripts from the BamHI-A region (BARF0), and latent membrane protein 2A (LMP2A). In most BL cell lines, latency is converted from type I to type III in serial cultures, in which cells express all the EBNAs (types 1, 2, 3A, 3B, 3C, and LP ), LMPs (types 1, 2A, and 2B), EBERs, and BARF0 [9]. Akata cells were originally 100% EBV positive, but after about 2 years of  in vitro cultivation, a fraction of cells became EBV negative. We could successfully isolate EBV-positive and -negative subclones by the limiting dilution method [12]. Comparison of EBV-positive and -negative Akata cell clones with identical cellular backgrounds enabled us to determine whether any phenotypic differences in cells were due to EBV. Using this system, we verified that, in Akata cells, EBV was necessary for the malignant phenotype, resistance to apoptosis, and upregulated expression of bcl-2 oncoprotein [7, 12]. We also demonstrated that EBER was responsible for these phenotypes [6]. Similar results have been reported by other groups [2, 10, 11, 14]. We further demonstrated that EBER induces expression of cellular interleukin-10 (IL-10) in BL cells, including Akata and Mutu cells, and that the induced IL-10 acts as an autocrine growth factor for BL [5]. These results clearly demonstrate that EBER is very important for the growth and malignant conversion of BL cells.

EBV is maintained as an episome in EBV-infected cells. It has been reported that the system of plasmid maintenance operated by binding of EBNA1 to the replication origin of the plasmid that contains EBV (oriP) [15] is not perfect, and 4% of cells per generation lose EBV plasmids [4]. If so, theoretically, the EBV-negative population should increase during cultivation, although we have not seen such a phenomenon in most EBV-infected cultures, with a few exceptions, including Akata cell culture. The most probable explanation is that EBV-infected cell-lines depend on the presence of EBV for their survival. Accumulation of mutations of cellular genes during cultivation of Akata cells made some fraction of the cells independent of EBV under ordinary culture conditions, and thus, we could isolate EBV-negative subclones.

Based on this background, in this study, we examined whether EBER could replace the role of EBV in Akata cells. As described above, EBV-negative cells appeared in an Akata cell culture that had been continuously cultivated in vitro for about 2 years. From that culture, we could isolate EBV-negative subclones by the limiting dilution method. In contrast, an early culture of the Akata cell line, referred to as Akata-EC, maintained the EBV plasmid stably. Several attempts failed to isolate EBV-negative subclones from Akata-EC cells by limited dilution. These results suggest that survival of Akata-EC cells depends on EBV infection. Therefore, we used Akata-EC cells to test whether EBER could be subsituted for EBV.

EBER1 and -2 open reading frames are located at bp 6628 to 6796 and 6958 to 7129, respectively, on the EcoR1 K fragment of Akata EBV DNA, which corresponds to the EcoR1-J fragment of B95-8 EBV DNA [1]. Since the plasmid that contained a single copy of EBER could not induce levels of EBER expression in transfected cells equivalent to those in EBV-infected cells, we used the plasmid that contained 10 tandem repeats of the EBER1 and -2 subfragment (Sac1-EcoR1 fragment, bp 6297 to 7325) from the EcoR1-K fragment of Akata EBV DNA and a neomycin resistance (Neor) gene driven by the simian virus 40 promoter [7]. By electroporation, Akata-EC cells (5 x 106) were transfected with either the EBER-containing plasmid or the control plasmid (pNeor), which contained a neomycin resistance gene driven by the simian virus 40 promoter. After 2 days of transfection, cells were transferred to 96-well, flat-bottom plates at 5,000 cells per well in complete culture medium containing 1.5 mg of G418 per ml (GIBCO). Half of the medium was changed every 6 days until colonies emerged. Eight Neor-transfected  clones and 14 EBER-trasfected clones were cultured in selective medium for 1 year.

These clones were examined by Southern blot analysis for the existence of EBV and EBER plasmids. Ten micrograms of cellular DNA was digested with the EcoR1 restriction enzyme, separated in a 0.8% agarose gel, blotted onto Hybond N+ nylon membranes (Amersham), and hybridized with a 32P-labeled EBER probe.

FIG. 1. (A) Detection of DNA from the plasmid and transfected EBER DNA in Akata-EC cell clones. Akata-EC cells were transfected with the EBER plasmid carrying the neomycin resistance gene or a Neor plasmid (as a control) and cultured in selective medium containing 1.5 mg of G418 per ml. G418-resistant cell clones were isolated and continuously cultivated for 1 year. Cellular DNA (10 mg) was isolated from each cell clone at 3 months and 1 year of culture, digested with EcoR1 restriction endonuclease, blotted onto a nylon membrane, and hybridized with a 32P-labeled EBER probe. The 3.0-kbp band is DNA from the EBV plasmid, and the 1.1-kbp is transfected EBER DNA. Photos of ethidium bromide-stained agarose gel are also shown. (B) EBNA expression in EBER- and Neor-transfected Akata-EC cell clones. EBNA was stained in the anticomplement immunofluorescence assay.


As shown in Fig. 1A, all Neor-transfected Akata-EC clones maintained the EBV-containing plasmid, as illustrated by the presence of a 3.0-kb band. The intensities of the bands were similar among the clones and did not change even after 1 year of culture, indicating that the EBV plasmid is stably maintained in Akata-EC cells. In contrast, the intensity of the 3.0-kb band varied considerably among EBER-transfected clones. The 3.0-kb band was undetectable in one clone (clone 3) after 3 months of culture and in four clones (clones 3, 8, 9, and 13) after 1 year of culture.

Expression of EBNA in Neor-, or EBER-transfected Akata-EC clones was examined by immunofluorescence assay. Cell smears on glass slides were fixed in acetone-methanol (1:1) for 2 min and sequentially incubated with a standard EBNA-positive human serum containing complement and fuorescein iso-thiocyanate-conjugated antihuman C3c (Dako). The results are summarized in Table 1.
 
TABLE 1. Percentage of EBNA-positive cells in Neor- and EBER-transfected Akata-EC clonesa
Akata-EC Cell Clone % EBNA-postive in cell 
clones cultured for: 		 .
. 3 months 1 year
Neor transfected . .
   1 >95 >95
   2 >95 >95
   3 >95 >95
   4 >95 >95
   5 >95 >95
   6 >95 >95
   7 >95 >95
   8 >95 >95
. . .
EBER transfected . .
   1   90   80
   2 >95   95
   3     0     0
   4   95 >95
   5   30   20
   6   90   45
   7   80   40
   8   20     0
   9   50     1
   10 >95   95
   11 >95   75
   12 >95   90
   13   60     0
   14   40     5

a EBNA was stained by the anticomplement immunofluorescence assay. More than 1,000 cells were examined to estimate the percentage of EBNA-positive cells.


All Neor-transfected clones were virtually 100% positive for EBNA at any culture time. In contrast, the frequency of EBNA-positive cells varied considerably from 0% to >95% among EBER-transfected clones. There was a good correlation between the intensity of the band of the EBV plasmid in Southern blot analysis (Fig. 1A) and the frequency of EBNA-postive cells. Figure 1B shows the EBNA staining of representative clones. In summary, these results indicate that transfection of the EBER gene causes a loss or decrease of EBV plasmid in Akata-EC cells.

Although about half of EBER-transfected Akata-EC clones lost the EBV genome, the rest retained the virus like before after a year of culture. This might have been due to the low expression of EBER from the transfected EBER-containing plasmid. However, we cannot determine EBER expression from the transfected EBER plasmid in EBV-positive cells, becuase we cannot discriminate EBER transcribed from the endogenous EBV genome from that transcribed from the transfected EBER plasmid. Therefore, we transfected EBER plasmid into EBV-negative Akata cells. After transfection, cells were transferred into 96-well plates and incubated in selective medium containing 1 mg of G418 per ml. Twenty-seven drug-resistant cell clones were randomly selected and examined for the expression of EBER by reverse transcription-PCR (RT-PCR), as described previously [6].

FIG. 2. EBER expression in EBER-transfected Akata cell clones. EBER expression was determined by the RT-PCR method as described previously [6]. (A) Determination of EBER expression by using different amounts of cDNA as templates. cDNA (100 ng) from EBV-positive Akata cells was diluted twofold and used as a template for PCR. (B) EBER expression in EBER-transfected EBV-negative Akata cell clones. One hundred nanograms of cDNA was used as a template for PCR. (C) EBER expression in EBER-tansfected Akata-EC cell clones from which the EBV plasmid was lost. One hundred nanograms of cDNA was used as a template for PCR.


As shown in Fig. 2, six clones expressed EBER at a level equivalent to or higher than that of EBV-positive Akata cells, but in nine clones, EBER was undetectable, and the remaining clones expressed it at a lower level. In contrast, four Akata-EC clones (clones 3, 8, 9, and 13), from which EBV plasmid was lost after EBER transfection (Table 1), expressed EBER at a level equivalent to that of EBV-positive Akata cells.These results suggested that clonal variation in EBER expression could explain why about half of EBER-transfected Akata-EC cell clones retained EBV plasmid.

Besides Akata cells, we examined other type 1 BL lines, such as Mutu I [3], Oku I. Sav I, and Kem I, to see whether EBER could replace the EBV plasmid in these cell lines.

FIG. 3 Detection of DNA from EBV plasmid and transfected EBER DNA in Mutu I cell clones. Akata-EC cells were transfected with EBER-containing plasmid carrying the neomycin resistance gene or a Neor plasmid (as a control) and cultured in selective medium containing 1.5 mg of G418 per ml. G418-resistant cell clones were isolated and cultivated for 1 year. Cellular DNA (10 mg) was isolated from each cell clone at 1 year of culture, digested with EcoR1 restriction endonuclease, blotted onto a nylon membrane, and hybriized with 32P-labeled EBER probe.The 3.0-kbp band is DNA from the EBV plasmid, and the 1.1-kbp band is transfected EBER DNA. Photos of ethidium bromide-stained agarose gel are also shown. (B) EBNA expression in EBER-transfected and Neor-transfected Mutu I cell clones. EBNA was stained in the anticmpiment immunofluorescence assay.


Southern blot analysis indicated that the level of the EBV plasmid decreased in four of the nine Mutu I cell clones that were transfected with the EBER plasmid (Fig. 3A). The immunofluorescence asay indicated that in at least one clone (clone 3), EBNA positivity decreased to 40%, while all Neor-transfected clones retained 100% EBNA positivity (Fig. 3B). On the other hand, in the Oku I, Sav I, and Kem I cell lines, transfection of EBER plasmid did not influence the copy number of the EBV plasmid (data not shown).

Finally, we studied whether genome loss relected some inhibitory effect of EBER on oriP function. EBV-negative Aakata cells were transfected with a plasmid, EBO, containing oriP, the EBNA1 gene, and the Neor gene [8] and maintained in medium containg G418. G418-resistant cell clones were then isolated. These cell clones carrying the plasmid containing oriP stably were transfected with the EBER plasmid or the Neor gene. Seven days after transfection, cells were harvested for detection of oriP plasmid DNA.

FIG. 4. Effect of EBER expression on retention of oriP-containing plasmid. EBV-negative Akata cell clones stably carrying the oriP-containing plasmid EBO were transfected with the Neor or EBER plasmid. Seven days after transfection, cells were harvested for detection of oriP DNA. Cellular DNA (10 mg) was digested with HindIII restriction endonuclease, blotted onto a nylon membrane, and hybridized with a 32P-labeled EBO probe.


Southern blot analysis indicated that there was no difference in the intensities of the oriP DNA bands between EBER- and Neor-transfected cells (Fig. 4). The results indicate that EBER does not influence retention of  the oriP-containing plasmid.

In the present study we have demonstrated that EBER plasmid could replace the EBV plasmid in at least two type I BL cell lines, Akata and Mutu. These results are consistent with our previous reports that EBER is important for the growth and malignant phenotypes of type I BL cell lines [5, 6]. However, we failed to replace EBV plasmid with the EBER plasmid in the other three type I BL cell lines. These results suggest that other EBV gene products play a role in the growth of BL cells. 


We thank J. Sample for the Mutu I, Oku I, Kem I, and Sav I cell lines; K. Adachi for technical assistance; and K. Inoue for help with preparing the manuscript.

This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan.

References:

1. Baer RA, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, Hutful C, Hudson GS, Satchwell SC, Seguin C, Tuffnell PS, and Barrell BG. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London) 310: 207-211.

2. Chodosh J, Holder VP, Gan Y, Belgaumi A, Sample J, and Sixbey JW. 1998. Eradication of latent Epstein-Barr virus by hydoxyurea alters the growth-transformed cell phenotype. J. Infect. Dis. 177: 1194-1201.

3. Gregory CD, Rowe M, and Rickinson AB. 1990. Different Epstein-Barr virus-B cell interactions in phenotypically distinct clones of a Burkitt's lymphoma cell line. J. Gen. Virol. 71: 1481-1495.

4. Kirchmaier AL, and Sugden B. 1995. Plasmid maintenance of derivatives of oriP of Epstein-Barr virus. J. Virol. 69: 1280-1283.

5. Kitagawa N, Goto M, Kurozumi K, Maruo S, Fukayama M, Naoe T, Yasukawa M, Hino K, Suzuki T, Todo S, and Takada K. 2000. Epstein-Barr virus-encoded Poly (A)- RNA supports Burkitt's lymphoma growth through interleukin-10 induction. EMBO J. 19: 6742-6750.

6. Komano J, Maruo S, Kurozumi K, Oda T, and Takada K. 1999. Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt's lymphoma cell line Akata. J. Virol. 73: 9827-9831.

7. Komano J, Sugiura M, and Takada K. 1998. Epstein-Barr virus contributes to the malignant phenotype and to apoptosis in Burkitt's lymphoma cell line Akata. J. Virol. 72: 9150-9156.

8. Margolskee RF, Kavathas P, and Berg P. 1988. Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. Mol. Cell Biol. 8: 2837-2847.

9. Rickinson AB, and Kieff E. 1996. Epstein-Barr virus, p. 2397-2446. In Fields BN, Knipe DM, and Howley PM, (ed.), Fields virology, 3rd ed. Lippencott-Raven, Philadelphia, Pa.

10. Ruf  IK, Rhyne PW, Yang C, Borza CM, Hutt-Fletcher LM, Cleveland JL, and Sample JT. 1999. Epstein-Barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt's lymphoma. Mol. Cell Biol. 19: 1651-1660.

11. Ruf  IK, Rhyne PW, Yang C, Cleveland JL, and Sample JT. 2000. Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt's lymphoma cells independently of an effect on apoptosis. J. Virol. 74: 10223-10228.

12. Shimizu N, Tanabe-Tochikura A, Kuroiwa Y, and Takada K. 1994. Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV. J. Virol. 68: 6069-6073.

13. Takada K, Horinouchi K, Ono Y, Aya T, Osato T, Takahashi M, and Hayasaka S. 1991. An Epstein-Barr virus-producer line Akata: establishment of the cell line and analysis of viral DNA. Virus Genes 5: 147-156.

14. Yamamoto N, Takizawa T, Iwanaga Y, Shimizu N, and Yamamoto N. 2000. Malignant transformation of B lymphoma cell line BJAB by Epstein-Barr virus-encoded small RNAs. FEBS Lett. 484: 153-158.

15. Yates JL, Warren N, Reisman D, and Sugden B. 1984. A cis-acting element from the Epstein-Barr virus genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. Natl. Acad. Sci. USA, 81: 3806-3810.



NCBI Nucleotide Data Base Entry, "EBER 1 small RNA":
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=16326314&dopt=GenBank

1: AB065135. Human herpesvirus...[gi:16326314]   Related Sequences,  PubMed, Taxonomy

LOCUS             AB065135      167 bp    DNA             VRL       23-OCT-2001
DEFINITION  Human herpesvirus 4 gene for EBER 1 small RNA, complete sequence.
ACCESSION   AB065135
VERSION        AB065135.1  GI:16326314
KEYWORDS    .
SOURCE          Human herpesvirus 4 (strain:Akata) DNA.
  ORGANISM  Human herpesvirus 4
                           Viruses; dsDNA viruses, no RNA stage; Herpesviridae;
                           Gammaherpesvirinae; Lymphocryptovirus.
REFERENCE   1  (bases 1 to 167)
  AUTHORS      Maruo,S., Nanbo,A. and Takada,K.
  TITLE              Replacement of the epstein-barr virus plasmid with the eber plasmid
                            in burkitt's lymphoma cells
  JOURNAL       J. Virol. 75 (20), 9977-9982 (2001)
  MEDLINE       21443992
REFERENCE    2  (bases 1 to 167)
  AUTHORS       Takada,K., Maruo,S. and Nanbo,A.
  TITLE              Direct Submission
  JOURNAL        Submitted (09-JUL-2001) Asuka Nanbo, Institute for Genetic
                             Medicine, Department of Tumor Virology; N15 W4, Kita-ku, Sapporo,
                             Hokkaido 060-8638, Japan.  E-mail:   nanboq@med.hokudai.ac.jp
                             Tel:  +81-11-706-5072,   Fax:  +81-11-717-1128
FEATURES                Location/Qualifiers
     source                     1..167
                                     /organism="Human herpesvirus 4"
                                     /strain="Akata"
                                     /db_xref="taxon:10376"
                                      /note="common name:Epstein-Barr virus"
     misc_RNA              1..167
                                      /product="EBER 1 small RNA"
BASE COUNT       23 a     43 c     55 g     46 t
ORIGIN
          1 aggacctacg ctgccctaga ggttttgcta gggaggagac gtgtgtggct gtagccaccc
        61 gtcccgggta caagtcccgg gtggtgagga cggtgtctgt ggttgtcttc ccagactctg
      121 ctttctgccg tcttcggtca agtaccagct ggtggtccgc atgtttt
//
Revised: October 24, 2001.


Additional References:

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b. Takada K, and Nanbo A, "The Role of EBERs in Oncogenesis", Seminars in Cancer Biology, vol. 11, no. 6, pp. 461-467 (December, 2001).

1a. Su Z, Peluso M, Raffegerst S, Schendel D, Roskrow M, "Dendritic Cells Transfected with LMP2a RNA Induce CD4+ LMP2a-Specific CTLs Which Kill via a Fas-Independent Mechanism", 43rd Annual Meeting, American Society of Hematology, Orlando, Florida, December 7-11, 2001, paper # 979.

1b. Maggio EM, van den Berg A, Visser L, Diepstra A, Emmens R, and Poppema S, "Common and Differential Chemokine Expression Patterns in RS Cells of NLP, EBV Positive and Negative Classical Hodgkin Lymphomas", 43rd Annual Meeting, American Society of Hematology, Orlando Florida, December 7-11, 2001, paper # 2506.

2. Kang M-S, Hung SC, and Kieff E, "Epstein-Barr Virus Nuclear Antigen 1 Activates Transcription from Episomal but Not Integrated DNA and Does Not Alter Lymphocyte Growth", Proc. Natl. Acad. Sci. USA, vol. 98, no. 26, pp. 15233-15238, (December 18, 2001). Published online before print December 4, 2001:
http://www.pnas.org/cgi/doi/10.1073/pnas.211556598

3. Ansardi DC, Porter DC, Jackson CA, Gillespie GY, and Morrow CD, "RNA Replicons Derived from Poliovirus are Directly Oncolytic for Human Tumor Cells of Diverse Origins",  Cancer Res. vol. 61, no. 23, pp. 8470-8479 (December 1, 2001).

4. Ruf IK, Rhyne PW, Yang C, Cleveland JL, and Sample JT, "Epstein-Barr Virus Small RNAs Potentiate Tumorigenicity of Burkitt Lymphoma Cells Independently of an Effect on Apoptosis", J. Virol. vol. 74, no. 21, pp. 10223-10228 (November, 2000).

5. Chapman ALN, Rickinson AB, Thomas WA, Jarrett RF, Crocker J, and Lee SP, " Epstein-Barr Virus-Specific T Lymphocyte Responses in the Blood and Tumor Site of Hodgkin's Disease Patients: Implications for a T-Cell Based Therapy", Cancer Research vol. 61, no. 16, pp. 6219-6226 (August 15, 2001).

6. Eddy SR, "Non-Coding RNA Genes and the Modern RNA World", Nature Reviews Genetics vol. 2, no. 12, pp. 919-929 (December, 2001).

7. Frenster JH, "Activation of DNA Transcription Within Repressed Chromatin", 14th John Innes Symposium, September 5-8, 2001.

8. Frenster JH, Papalian MM, Masek MA and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", J. National Cancer Institute, vol. 63, pp. 331-335  (Aug. 1979).

9. Frenster JH, "Selective Control of DNA Helix Openings During Gene Regulation", Cancer Research vol. 36, pp. 3394-3398 (September, 1976).

10. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clin. Cancer Res. vol. 5, supp. 1, p. 3855s, (624) (November, 1999).

11. Mattsson K,  Kiss C,  Platt GM,  Simpson GR,  Kashuba E,  Klein G,  Schulz TF,  and Szekely L, "Latent Nuclear Antigen of Kaposi's Sarcoma Herpesvirus/Human Herpesvirus-8 Induces and Relocates RING3 to Nuclear Heterochromatin Regions',  J. Gen. Virol. vol. 83, no. 1, pp. 179-188 (January, 2002).

12. Vinogradova T, Leppik L, Kalinina E, Zhilidov P, Grzeschik K-H, and Sverdlov E, "Selective Differential Display of RNAs Containing Interspersed Repeats: Analysis of Changes in the Transcription of HERV-K LTRs in Germ Cell Tumors", Molecular Genetics and Genomics vol. 266, no. 5, pp. 796 805 (January, 2002).

13. Frenster JH, "Uni-polar Clustering of Lymphocyte DNA Templates Toward Neoplastic Target Cells Within Hodgkin's Disease Lymph Nodes". Proc. Am. Assoc. Cancer Res. vol. 43, p. 1134 (March, 2002).

14. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA", (42-Slide Presentation).

15. Carella AM, "Stem Cell Transplantation for Hodgkin's Disease: A Review of the Literature", Clinical Lymphoma, vol. 2, no. 4, pp. 212-221 (March, 2002).
 


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