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Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and... Background: The diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members. Results: MMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own. Conclusion: The presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice. Background an association of the betaretrovirus mouse mammary Three members of the same family, father, mother, and tumor virus (MMTV) with a certain percentage of human daughter, were diagnosed with carcinoma of the breast breast tumors [1-5]. In addition we identified MMTV-like with axillary nodal metastases. The father was the first to DNA sequences in both breast tumor tissue and non- be diagnosed at the age of 79 in 1963. The mother and Hodgkin's lymphoma tissue of eight patients diagnosed daughter were each diagnosed six years later in 1969 at the with both diseases and in the lymphoma tissue of three ages of 82 and 56 respectively. All three family members patients diagnosed with only non-Hodgkin's lymphoma had invasive carcinoma as shown in Figure 1. Published [6]. We and others have not detected MMTV in normal data from five laboratories including our own have shown human tissue [1,6,7]. In mice MMTV is the etiological Page 1 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 astat Figure 1 Hemia c breast tumor toxylin and eosin sta tissuei in ned lymph slides nod of fo e rmalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue and met- Hematoxylin and eosin stained slides of formalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue and metastatic breast tumor tissue in lymph node. Panel A (father): metastatic ductal carcinoma of breast in axillary lymph node. The tumor is almost completely replacing the normal tissue in this 2-cm node. Note the rim of residual subcapsular lymphoid tissue. Panel B (mother): invasive moderately differentiated ductal carcinoma of breast. Note the prominent lymphocytic response. Panel C (daughter): invasive and in situ lobular carcinoma of breast. Only a portion of the round edge of a lobule containing lobular carcinoma in situ is seen here. Magnification is 200X. Page 2 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 agent responsible for the development of breast tumors as Presence of MMTV-like env sequences in breast cancer well as certain B-and T-cell lymphomas [8-10]. In this DNA from malignant tissue of each family member was study we investigated the presence of MMTV-like DNA amplified using single round PCR with primers specific sequences in three family members each of whom had for a 250 basepair (bp) region of the MMTV env gene and been diagnosed with breast cancer. We have detected the not found in any human endogenous retroviral sequences presence of both the MMTV-like envelope (env) and long i.e HERV-Ks [1,4,11,12]. Figure 2A represents the ethid- terminal repeat (LTR) gene sequences in all three patients. ium bromide-stained agarose gel electrophoresis of an MMTV env primed PCR from each of the three family members and 2B is the hybridized Southern blot [13]. Results Slides of tumor tissue Lanes 2, 3, and 4 in both Figure 2A and 2B represent the Figure 1A–C represents the hematoxylin and eosin stained amplified DNA from the daughter, mother, and father slides from the formalin-fixed paraffin-embedded tissue respectively. Lane 1 containing no template DNA and sample blocks obtained from each of the three family lanes 5, 6, and 7 containing normal breast tissue DNA members. Samples blocks from mother and daughter con- from three separate individuals represent negative con- tained malignant tissue from their respective breast trols for sample contamination. Positive hybridization tumors. Sample blocks from the father were from a lymph results with the radiolabled internal 23-mer oligonucle- node that contained metastatic breast cancer. otide probe that contained MMTV-env gene sequences A Figure 2 mplification of 250 bp of MMTV-like env gene and 630 bp of MMTV-like LTR gene by PCR Amplification of 250 bp of MMTV-like env gene and 630 bp of MMTV-like LTR gene by PCR. A, Ethidium bromide stained 1% agarose gel electrophoresis of amplified MMTV-like env sequences; B, Southern blot [13] hybridization of A using 5' P end-labeled 23-mer probe. Lanes 2, 3 and 4 represent the amplified DNA from the daughter, mother and father respec- tively. C, Ethidium bromide stained 1.8 % agarose gel electrophoresis of amplified MMTV-like LTR sequences; D, Southern blot [13] hybridization of C using 5' P end-labeled 20 mer probe. Lanes 2, 3, and 4 represent amplified DNA from mother, daugh- ter and father respectively. In Panels A-D, Lane 1 containing no template DNA and lanes 5, 6, and 7 containing normal breast tissue DNA from three separate individuals represent negative controls for sample contamination. M is the molecular weight marker ΦX174 RF DNA cut with the restriction enzyme HaeIII. Page 3 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 indicated that this MMTV-specific sequence was present in change. Previously we had identified an identical single the amplified 250-bp fragment and that the bands in the base change in the MMTV env sequences that were present agarose gel were MMTV specific. in a number of breast tumors and all non-Hodgkin's lym- phomas [6]. Presence of MMTV-like LTR sequences in breast cancer DNA from the three afflicted family members was ampli- Cloning and sequencing of amplified MMTV-like LTR ORF sequences from human breast cancer fied by single round PCR with primers specific for a 630- bp region within the MMTV LTR open reading frame The amplified 630 bp MMTV-like LTR ORF sequences (ORF) that codes for the MMTV superantigen (sag) gene present in the primary and metastatic breast tumor tissue [14,15] and that is not present in HERV-K sequences were cloned using the Invitrogen TOPO TA Cloning kit for [6,11,12,16]. Figure 2C represents the ethidium bromide- Sequencing. A total of 12 clones (4 for each family mem- stained agarose gel electrophoresis of the MMTV LTR ber) were sequenced. The U3 region of the MMTV LTR primed PCR of the three family members and Figure 2D contains the open reading frame (ORF) that encodes the represents the hybridized Southern blot [13]. Lanes 2, 3, glycoprotein superantigen (sag) that is present in all exog- and 4 in both Figure 2C and 2D represent amplified DNA enous and endogenous MMTV viral sequences [14,15]. from the mother, daughter, and father respectively. Lane 1 Although highly conserved, the MMTV sag sequences are containing no template DNA and lanes 5, 6, and 7 con- not identical with approximately 35% of the total varia- taining normal breast tissue DNA from three separate tion clustered at the hypervariable COOH terminus. This individuals represent our negative controls for sample variation present in the COOH terminus of the MMTV sag contamination. Positive hybridization with the radiola- gene is specific to each MMTV provirus. beled internal 20-bp oligonucleotide probe that con- tained MMTV-LTR gene sequences indicated that this In the conserved regions of the LTR ORF all 12 clones MMTV-specific sequence was present in the amplified shared 96–98 percent homology with numerous strains of 630-bp fragment and that the bands in the agarose gel MMTV and with each other. However, within the highly were specific for MMTV LTR sequences. variable COOH terminus of the sag gene all of the 12 iso- lated clones were either identical or nearly identical to Cloning and sequencing of amplified MMTV-like env from either the MMTV proviral sequence Mtv-8 [20,21] or Mtv- human breast cancer 1 [22,23] as shown in Figure 4. Each of the three family The amplified MMTV env gene-specific 250-bp sequences members contained COOH-terminal sag sequences that present in the primary and metastatic breast tumor tissue were identical or nearly identical (one or two base differ- were cloned using the Invitrogen TOPO TA Cloning kit for ences) to one of these two proviral sequences. Sequencing. A total of 12 clones (4 for each family mem- ber) were sequenced. The sequences of the 12 amplified To analyze relationships between viral strains, phyloge- fragments were shown to be 98.0% – 99.6 percent homol- netic trees have been constructed on the basis of align- ogous to the GR, C3H and BR6 mouse strains of MMTV in ments of LTR ORF sequences [24]. Phylogenetic analysis this 250-bp region of the MMTV env gene. Figure 3 shows of the entire 630 bp LTR ORF sequences isolated from the comparison of the sequences of the 12 clones to the each of the three family members diagnosed with breast three strains of MMTV [17-19] and to each other in this cancer is shown in Figure 5. All 12 of the human MMTV- region of the MMTV env gene. like LTR ORF sag clones segregated to two branches of the MMTV phylogenetic tree, Mtv-8 [20,21] and Mtv-1 As shown in Figure 3 the sequenced env clones fell into 7 [22,23], and did not form a branch of their own. Moreo- classes that differed from one another. However no one ver, clones isolated from the same family member segre- env clone differed from any other by more than 4 base gated to these two separate branches. Interestingly, all of changes either within the same family member or the prior MMTV-like LTR isolates from breast cancer between family members. One env clone each from the [6,16,25], non-Hodgkin's lymphoma [5], and primary mother (M1), father (F2), and daughter (D2) were identi- biliary cirrhosis [26,27] patients also associated with these cal to each other. Two env clones from the father (F3, F4) two branches of the MMTV phylogenetic tree. were identical to each other, one daughter env clone (D1) and one father env clone (F1) were the same, and two env Discussion clones from the mother (M2, M3) were the same. Each To our knowledge, this is the first report of breast cancer family member however, mother (M1, M4), father (F2, in father, mother, and daughter. We acknowledge how- F3, F4), and daughter (D2, D3), each contained env clones ever limitations of our study due to the quality of the DNA that included a single base change mutation denoted by of the archival formalin fixed paraffin samples on which the arrow in Figure 3. Each family member also contained this study is based. Limitations include our inability to env clones (M2, M3, F1, D1, D4) without this single base determine the mutation status of the BRCA1 and BRCA2 Page 4 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 GR/C3H TACATCTGCC TGTGTTACTT ACCCTTATGC CATATTATTA GGATTACCTC BR/6 ------------------- ------------------ ------------------- ------------------- ------------------- ALL 12 -------------------- ------------------ -------------------- -------------------- ------------------- GR/C3H AGCTAATAGA TATAGAGAAA AGAGGATCTA CTTTTCATAT TTCCTGTTCT BR/6 -----T-------------- --------------------- -------------------- ------------------- ------------------- 9 of 12 --------------------- --------------------- -------------------- ------------------- ------------------- F3,F4 -------------------- --------------------- -------------------- ------------------- -------T----------- D3 -----T-------------- --------------------- -------------------- ------------------- -------------------- 2 ↓↓↓↓ GR/C3H TCTTGTAGAT TGACTAATTG TTTAGATTCT TCTGCCTACG ACTATGCAGC BR/6 ------------------- ------------------- -----------C------ ------------------- --------------------- F3,F4 ------------------- ------------------- ------------------- ------------------- -----------A-------- D3 C------------------ C------------------ -------------------- ------------------- -----------A-------- M1,D2.F2 ------------------- ------------------- -------------------- ------------------- -----------A-------- M4 ------------------- ------------------- -------------------- ------------------- -----------A-------- D1,F1 ------------------- -------------------- -------------------- ------------------- ---------------------- M3,M2 -------------------- --------------------- --------------------- ------------------- ---------------------- D4 ------------------- --------------------- ------------C----- -------------------- ---------------------- GR/C3H GATCATAGTC AAGAGGCCGC CATACGTGCT GCTACCTGTA GATATTGGTG BR/6 -------------------- -------------------- --------T---------- ------------------- ------------------- F3,F4 --------------------- -------------------- -------------------- ------------------- -------------------- D3 --G----------------- -------------------- --------------------- ------------------- -------------------- M1,D2, F2-------------------- -------------------- --------------------- ------------------- -------------------- M4 ------------------- --------------------- ---------T---------- ----------------C-- ------------------- D1,F1 --------------------- --------------------- ---------T---------- -------------------- ------------------- M3,M2 --------------------- --------------------- ---------T---------- ----------------C-- ------------------- D4 ------------------- -------------------- ---------T---------- -------------------- -------------------- GR/C3H ATGAACCATG GTTTGATGAT TCTGCCATTC AAACCTTTAG GTATGCCACA BR/6 --------------------- -------------------- ------------------- -------------------- --------------------- F3,F4 --------------------- -------------------- ------------------- -------------------- ---------------------- D3 --------------------- -------------------- -------------------- ------------------- ---------------------- M1,D2,F2--------------------- -------------------- -------------------- -------------------- ---------------------- M4 ---------------------- -------------------- -----C-------------- -------------------- --------------------- D1,F1 ---------------------- -------------------- --------------------- -------------------- ---------------------- M3,M2 ---------------------- -------------------- ----C-------------- -------------------- --------------------- D4 ---------------------- -------------------- -------------------- ------------------- --------------------- a ge Figure 3 Seq nne d daugh u of G ences of R ter , C3 and the 250 H a meta nd B bp PCR MMTV-like sta R6 s tic tbreast rains o tumor t f MMTenv Vissue in lymph gene producnode t ampl of father ified from compared with the DNA of pth rimary breast cancer e sequences in this r tissue of egion of the mother env Sequences of the 250 bp PCR MMTV-like env gene product amplified from the DNA of primary breast cancer tissue of mother and daughter and metastatic breast tumor tissue in lymph node of father compared with the sequences in this region of the env gene of GR, C3H and BR6 strains of MMTV. The numbers 1, 2 and 3 indicate the three locations where the BR6 strain differs from the GR and C3H strains in this region of the MMTV env gene [17,18,19]. The A and ↓ indicate the location at which the identical single base change described in the text occurs. The numbers 7656 and 7905 indicate the location of the MMTV 250 bp env gene sequence within the MMTV genome [17]. Clones are designated as M (mother), D (daughter), and F (father) followed by a number (1–4) denoting the order in which they were cloned. - denotes the same nucleotide. Page 5 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 a Seq m Figure 4 no d Mtv ther a uence h -8 [20,21] to the amp nd da omology of the highly vari ughter and meta lified highly var static br abl ea e CO st tum iaOH ble MMTV or tissue in -terminal sag regio lymp sequences clon n h node of the father of the MMTV ed from sag the primar gene of MMTV provir y breast tuuses Mtv-1 [22,23] mor tissue of the Sequence homology of the highly variable COOH-terminal region of the MMTV sag gene of MMTV proviruses Mtv-1 [22,23] and Mtv-8 [20,21] to the amplified highly variable MMTV sag sequences cloned from the primary breast tumor tissue of the mother and daughter and metastatic breast tumor tissue in lymph node of the father. Clones are named as M (mother), D (daughter), and F (father) followed by a dash and the letter s for sag and numbers (1–4) identifying the order in which they were cloned. - denotes the same nucleotide. Page 6 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 P cluster as Figure 5 hylogeneti two di c analsti ysn isct s of human br pecies east tumor MMTV-like LTR sequences showing that the human and mouse sequences do not Phylogenetic analysis of human breast tumor MMTV-like LTR sequences showing that the human and mouse sequences do not cluster as two distinct species. The 12 human MMTV-like LTR sequences from the three family mem- bers as well as the human sequences previously isolated from human breast tumors, non-Hodgkin's lymphomas, and primary biliary cirrhosis tissue, clustered with their murine counterparts. Boxes denote LTR sequences from mother (M-S1-4), daughter (D-S1-4), and father (F-S1-4). Previously published human isolates AF346815, AY325271, AF243039, AY652977, AY652968, AY652964, AY652975, AY652974, AY652967, AY652969, AY652973, from human breast tumors [6,16,27], AY652970, AY652976, AY652978, AY652965, AY652971, AY652966, AY652972 from human non-Hodgkin's lymphomas [6], and AF513913, AF513923, from human primary biliary cirrhosis patients [26,27]. The mouse sequences, JYG, FM, and SW21 from Asian mice that were used to root the tree, the endogenous MMTV proviral sequences Mtv-8, Mtv-1, and Mtv-6, and the exog- enous MMTV sequences BR6, HEJ, and C3H are bolded. Numbers on branches indicate percent frequencies of assortment in an individual branch after the bootstrap procedure (45) and indicate the robustness of branch assignments. Branch lengths are indicative of the number of nucleotide changes to individual branch points (see scale bar). Page 7 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 genes in these patients [28]. However the late age at which The 630 bp MMTV-like DNA that we have amplified using these breast tumors developed argues against their being primers specific for regions of the ORF of the MMTV LTR caused by mutations in the BRCA1 or BRCA2 genes. codes for the superantigen (sag) gene of MMTV [14,15]. Quantity and quality of the archival DNA also limited us Although highly conserved, the MMTV sag sequences are in that we were able to successfully amplify only parts of not identical with approximately 35 percent of the total two regions, the env and LTR, of the MMTV-like DNA variation clustered at the hypervariable COOH terminus. genome and were not able to detect the presence of inte- This variation present at the COOH terminus of the grated MMTV-like viral sequences in the cellular genome. MMTV sag gene is specific to each MMTV strain. Each of our cloned sag sequences from each family member was Breast cancer in husband and wife may not be as uncom- identical or nearly identical to either the MMTV Mtv-8 mon as generally thought. At least ten couples have been [20,21] or Mtv-1 [22,23] proviral sequence. In a previous previously reported since 1975 [29-33]. Russ and Scanlon publication [6] we have shown that the cloned sag [31] reporting on eleven married couples having histolog- sequences present in both the breast tumor tissue and ically identical neoplasms, including three couples with non-Hodgkin's lymphoma tissue of eight patients diag- breast cancer, noted that the clinical course of the disease nosed with both malignancies also contained sag tended to be similar in both husband and wife, and that sequences that were identical or nearly identical to MMTV husband and wife were diagnosed usually within approx- proviral sequences Mtv-1 and Mtv-8. Similar sequences imately five years of each other. Both observations apply isolated from human breast tumors by others also con- to our family. Some studies however have not shown an tained sag sequences with identity to the Mtv-1 and Mtv-8 increased incidence of breast cancer in wives of men with proviral sequences [16,25]. Also, MMTV-like sag that neoplasm [33-35]. However, Russ and Scanlon noted sequences isolated from patients diagnosed with primary that the tumors they observed in husband and wife have biliary cirrhosis (PBC) also contained sag sequences with been suggested either experimentally or indirectly to have identity to the Mtv-1 and Mtv-8 proviral sequences viral relationships [31]. Lynch et al [36] suggested more [26,27]. Phylogenetic studies from our laboratory and than twenty years ago that a communicable agent might that of others [27] have shown that such sequences segre- play a role in the clustering of certain cancers in spouses. gate to specific branches of the MMTV phylogenetic tree and do not form a distinct branch of their own thus argu- The MMTV-like env sequences that we have detected in the ing for their being mouse derived and not human homo- mother, father, and daughter of this family are 98–99.6 logues of the mouse sequence [6,27]. This study, our percent identical to the GR, C3H and BR6 mouse strains previously published work [6], and that of others [26,27] of MMTV [17-19]. Recently the Env protein of MMTV also indicate that more than one viral strain of MMTV-like from the mouse has been shown to be capable of trans- DNA sequences may be present in the same individual. forming mouse and human mammary epithelial cells in vitro [37]. The MMTV Env protein contains an immunore- Other works that have shown that MMTV-like DNA ceptor tyrosine-based activation motif (ITAM) sequence sequences are for the most part not found in normal cells that appears to allow for its transformation ability [37]. [1-7,25] suggest that the presence of the virus in humans The MMTV-like env gene sequences that we have isolated may result from an exogenous infection. Very recently, a from the primary human breast tumor tissue and the met- virus closely related to the xenotropic murine leukemia astatic breast tumor tissue present in a lymph node in the viruses (MuLVs) has been detected in the tumor tissue of three family members studied in this report and in addi- a certain population of prostate cancer patients. The viral tional breast tumors previously reported and in non- sequence is not found in human genomic DNA thus indi- Hodgkin's lymphomas contain this ITAM sequence [1,6]. cating, as the authors discuss, an exogenous infection It is not yet known if the MMTV-like Env protein coded for [38,39]. The suggestion that MMTV exogenous infection by the MMTV-like DNA sequences which we and others can occur in humans is a highly controversial topic but is have detected in human breast tumors is capable of trans- becoming more plausible with two recent publications by formation. We have sequenced 4 env clones per family Indik et al [40,41]. In these papers the authors show that member for a total of 12 env clones. Seven of these 12 MMTV can infect human cells in vitro, make new MMTV, clones contain an identical single base substitution (Fig- and that this new MMTV can go on to infect other human ure 3, ↓) that we have detected in a number of additional cells. That the MMTV-like DNA sequences that we and breast tumors and in all the non-Hodgkin's lymphomas others have found in human breast tumors may be mouse we have analyzed. This base change of a G to A results in derived is also suggested by the findings that their sag the replacement of an alanine (GCA) with a threonine sequences segregate to arms of the MMTV phylogenetic (ACA) in these samples. Curiously, this single-base tree and do not form a separate branch of their own change occurs within the ITAM domain [37]. [6,26,27]. Additional epidemiological findings suggesting an exogenous infection from mice include the findings of Page 8 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 Stewart et al [42] in which it was shown that the incidence PCR of breast cancer was highest in countries in which the Conditions and primer sequences for MMTV env and LTR mouse strain mus domesticus resides, a mouse strain that gene amplification were those described by Wang et al [4] carries a large number of endogenous MMTV proviruses. and Liu et al [25] respectively. A reaction without template Curiously, a recent study from the Johns Hopkins Univer- DNA was routinely tested to detect possible contamina- sity Schools of Medicine and Public Health has reported tion of master mix components. Reactions with normal that airborne mouse allergen was found in 84 percent of breast DNA were done to rule out contamination of bedrooms of inner city homes in Baltimore and that the tumor samples. The PCR product was analyzed by electro- concentration of this mouse allergen may be similar to phoresis in 1.8 % agarose gels. ΦX174 RF DNA cut with those found in animal facilities [43]. We do not know the the restriction enzyme HaeIII was used as a marker to concentration of mouse allergen in the home of the family identify the size of the PCR products. in this study who lived in a wealthy Baltimore neighbor- hood. Hybridization PCR products were hybridized on Southern blots [13] Conclusion under stringent hybridization conditions to either a 23- The presence of MMTV-like DNA sequences in the malig- base pair (bp) probe specific for DNA sequences present nant tissues of all three family members who lived in the in exogenous MMTV env sequences but not present in same house for decades argues for the possibility of human endogenous retroviral sequence (HERV-K) DNA MMTV as a common environmental etiological agent. [1,4,11,12] or to a 20-bp probe specific for the MMTV LTR Phylogenetic data suggest that the MMTV-like sequences [6,16] and also not present in HERV-K DNA [11,12]. The are mouse and not human derived and that the ultimate 23-bp env probe, which extended from position 7822– reservoir of MMTV is most likely the mouse. Although the 7845 in the MMTV genome, and the 20-bp LTR probe, route by which these three family members came to be which extended from position 972–991 or 9545–9564 in infected with MMTV is unknown, the possibility exists the MMTV genome [17], were end-labeled with [ P]ATP that such infection may have resulted from an exposure to using the T4 kinase forward reaction (Invitrogen Life mice. Technologies, Inc., Carlsbad, CA). Stringent hybridization conditions were as described previously [1,11]. Methods Human tissue Cloning and sequencing of PCR products Formalin-fixed, paraffin-embedded tissue sample blocks Amplified DNA products were cloned directly from the of breast tumor tissue (mother and daughter) and meta- PCR tube using the TOPO TA Cloning kit for Sequencing static breast tumor in lymph node (father) were obtained (Invitrogen). DNA sequencing was performed by Gen- from the Johns Hopkins Medical Center, Baltimore, Mar- emed Synthesis (San Antonio, TX). The resulting yland, courtesy of Drs. Elizabeth Montgomery and Arlene sequences were compared to known published sequences Forastiere, under a protocol approved by the Institutional and to sequences in the Genbank. Review Board of Our Lady of Mercy Medical Center (OLMMC). Hematoxylin and eosin stained slides of each Phylogenetic analysis paraffin block were made and read to determine the loca- The LTR sequences obtained from cloned fragments after PCR amplification were compared to known exogenous tion of malignancy in each block. Blocks were shaved into 5 µm thick serial sections and two sections from each and endogenous MMTV sequences in the Genbank data- specimen were used for DNA extraction. DNA was iso- base. LTR sequences were aligned using DNAssist 2.0 soft- lated from the blocks using a microwave technique [44]. ware and analyzed using the phylogenetic Analysis Using Normal breast tissue samples were obtained from the Parsimony (PAUP 4.0b 10) program [45] as previously Pathology Department at OLMMC under a protocol described [6]. approved by the Institutional Review Board of OLMMC. DNA was extracted from normal breast tissue using the Authors' contributions QIAgen DNA Mini Kit (Qiagen Inc, Germantown, MD). PRE participated in the design of the study, carried out the To determine the quality of the isolated DNA from both molecular biology studies, and drafted the manuscript, paraffin blocks of tumor tissue and fresh normal breast AFRS carried out the phylogenetic studies and participated tissue, globin primers were used in PCR and the resulting in the drafting of the manuscript, PHW participated in the amplified products were electrophoresed in 1.8 % agarose design of the study and in the drafting of the manuscript. gels. Acknowledgements We thank Dr. Stanley Oiseth of the Department of Pathology at Our Lady of Mercy Medical Center (OLMMC) for supplying normal breast tissue and for his expertise in evaluating our paraffin blocks for study and for photos Page 9 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 of our tissue samples. We thank Drs. Elizabeth Montgomery and Arlene 20. Donehower LA, Fleurdelys B, Hager GL: Further evidence for the protein coding potential of the mouse mammary tumor Forastiere of the Johns Hopkins Medical Center for their contribution of virus long terminal repeat:Nucleotide sequence of an endog- paraffin blocks for this study. We also thank Marie Alice Lilazois and Julio enous proviral long terminal repeat. J Virol 1983, 45:941-949. Aviles in OLMMC Pathology for their excellent skill in preparing shavings of 21. Kennedy N, Knedlitschek G, Groner B, Hynes NE, Herrlich P, Michal- paraffin blocks for DNA isolation. This work was funded in part by the A.L. ides R, Van Ooyen AJ: Long terminal repeats of endogenous mouse mammary tumor virus contain a long open reading Levine Family Foundation, Inc. of Wayne, NJ. frame which extends into adjacent sequences. Nature(London) 1982, 295(5850):622-624. References 22. Pullen AM, Choi Y, Kushnir E, Kappler J, Marrack P: The open read- 1. 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Steinitz R, Katz L, Ben-Hur M: Male breast cancer in tumor virus-like env sequences in cancer and normal breast Israel:selected epidemiological aspects. Israel J Med Sci 1981, from the same individuals. Clin Cancer Res 2001, 7:283-284. 17:816-821. 8. Nandi S, McGrath C: Mammary neoplasia in mice. Adv Cancer Res 31. Russ JE, Scanlon EF: Identical cancers in husband and wife. Surg 1973, 17:353-414. Gynecol Obstet 1980, 150:664-666. 9. Michalides R, Wagenaar E, Hilkins J, Groner B, Hynes NE: Acquisi- 32. Wetchler BB, Simon B: Carcinoma of the breast occurring in a tion of proviral DNA of mouse mammary tumor virus in husband and wife:a brief communication. Mt Sinai J Med 1975, thymic leukemia cells from GR mice. J Virol 1982, 42:205-206. 43(3):819-829. 33. Friedman GD, Quesenberry CP Jr: Spousal concordance for can- 10. Dekaban GA, Ball JK: Integration of type B retroviral DNA in cer incidence. A cohort study. Cancer 2000, 86:2413-2419. virus-induced primary murine thymic lymphomas. 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Sunderland, Massachusetts:Sinauer Associates 2002. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 11 of 11 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

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Springer Journals
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Copyright © 2008 by Etkind et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-3-2
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18307792
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Abstract

Background: The diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members. Results: MMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own. Conclusion: The presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice. Background an association of the betaretrovirus mouse mammary Three members of the same family, father, mother, and tumor virus (MMTV) with a certain percentage of human daughter, were diagnosed with carcinoma of the breast breast tumors [1-5]. In addition we identified MMTV-like with axillary nodal metastases. The father was the first to DNA sequences in both breast tumor tissue and non- be diagnosed at the age of 79 in 1963. The mother and Hodgkin's lymphoma tissue of eight patients diagnosed daughter were each diagnosed six years later in 1969 at the with both diseases and in the lymphoma tissue of three ages of 82 and 56 respectively. All three family members patients diagnosed with only non-Hodgkin's lymphoma had invasive carcinoma as shown in Figure 1. Published [6]. We and others have not detected MMTV in normal data from five laboratories including our own have shown human tissue [1,6,7]. In mice MMTV is the etiological Page 1 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 astat Figure 1 Hemia c breast tumor toxylin and eosin sta tissuei in ned lymph slides nod of fo e rmalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue and met- Hematoxylin and eosin stained slides of formalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue and metastatic breast tumor tissue in lymph node. Panel A (father): metastatic ductal carcinoma of breast in axillary lymph node. The tumor is almost completely replacing the normal tissue in this 2-cm node. Note the rim of residual subcapsular lymphoid tissue. Panel B (mother): invasive moderately differentiated ductal carcinoma of breast. Note the prominent lymphocytic response. Panel C (daughter): invasive and in situ lobular carcinoma of breast. Only a portion of the round edge of a lobule containing lobular carcinoma in situ is seen here. Magnification is 200X. Page 2 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 agent responsible for the development of breast tumors as Presence of MMTV-like env sequences in breast cancer well as certain B-and T-cell lymphomas [8-10]. In this DNA from malignant tissue of each family member was study we investigated the presence of MMTV-like DNA amplified using single round PCR with primers specific sequences in three family members each of whom had for a 250 basepair (bp) region of the MMTV env gene and been diagnosed with breast cancer. We have detected the not found in any human endogenous retroviral sequences presence of both the MMTV-like envelope (env) and long i.e HERV-Ks [1,4,11,12]. Figure 2A represents the ethid- terminal repeat (LTR) gene sequences in all three patients. ium bromide-stained agarose gel electrophoresis of an MMTV env primed PCR from each of the three family members and 2B is the hybridized Southern blot [13]. Results Slides of tumor tissue Lanes 2, 3, and 4 in both Figure 2A and 2B represent the Figure 1A–C represents the hematoxylin and eosin stained amplified DNA from the daughter, mother, and father slides from the formalin-fixed paraffin-embedded tissue respectively. Lane 1 containing no template DNA and sample blocks obtained from each of the three family lanes 5, 6, and 7 containing normal breast tissue DNA members. Samples blocks from mother and daughter con- from three separate individuals represent negative con- tained malignant tissue from their respective breast trols for sample contamination. Positive hybridization tumors. Sample blocks from the father were from a lymph results with the radiolabled internal 23-mer oligonucle- node that contained metastatic breast cancer. otide probe that contained MMTV-env gene sequences A Figure 2 mplification of 250 bp of MMTV-like env gene and 630 bp of MMTV-like LTR gene by PCR Amplification of 250 bp of MMTV-like env gene and 630 bp of MMTV-like LTR gene by PCR. A, Ethidium bromide stained 1% agarose gel electrophoresis of amplified MMTV-like env sequences; B, Southern blot [13] hybridization of A using 5' P end-labeled 23-mer probe. Lanes 2, 3 and 4 represent the amplified DNA from the daughter, mother and father respec- tively. C, Ethidium bromide stained 1.8 % agarose gel electrophoresis of amplified MMTV-like LTR sequences; D, Southern blot [13] hybridization of C using 5' P end-labeled 20 mer probe. Lanes 2, 3, and 4 represent amplified DNA from mother, daugh- ter and father respectively. In Panels A-D, Lane 1 containing no template DNA and lanes 5, 6, and 7 containing normal breast tissue DNA from three separate individuals represent negative controls for sample contamination. M is the molecular weight marker ΦX174 RF DNA cut with the restriction enzyme HaeIII. Page 3 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 indicated that this MMTV-specific sequence was present in change. Previously we had identified an identical single the amplified 250-bp fragment and that the bands in the base change in the MMTV env sequences that were present agarose gel were MMTV specific. in a number of breast tumors and all non-Hodgkin's lym- phomas [6]. Presence of MMTV-like LTR sequences in breast cancer DNA from the three afflicted family members was ampli- Cloning and sequencing of amplified MMTV-like LTR ORF sequences from human breast cancer fied by single round PCR with primers specific for a 630- bp region within the MMTV LTR open reading frame The amplified 630 bp MMTV-like LTR ORF sequences (ORF) that codes for the MMTV superantigen (sag) gene present in the primary and metastatic breast tumor tissue [14,15] and that is not present in HERV-K sequences were cloned using the Invitrogen TOPO TA Cloning kit for [6,11,12,16]. Figure 2C represents the ethidium bromide- Sequencing. A total of 12 clones (4 for each family mem- stained agarose gel electrophoresis of the MMTV LTR ber) were sequenced. The U3 region of the MMTV LTR primed PCR of the three family members and Figure 2D contains the open reading frame (ORF) that encodes the represents the hybridized Southern blot [13]. Lanes 2, 3, glycoprotein superantigen (sag) that is present in all exog- and 4 in both Figure 2C and 2D represent amplified DNA enous and endogenous MMTV viral sequences [14,15]. from the mother, daughter, and father respectively. Lane 1 Although highly conserved, the MMTV sag sequences are containing no template DNA and lanes 5, 6, and 7 con- not identical with approximately 35% of the total varia- taining normal breast tissue DNA from three separate tion clustered at the hypervariable COOH terminus. This individuals represent our negative controls for sample variation present in the COOH terminus of the MMTV sag contamination. Positive hybridization with the radiola- gene is specific to each MMTV provirus. beled internal 20-bp oligonucleotide probe that con- tained MMTV-LTR gene sequences indicated that this In the conserved regions of the LTR ORF all 12 clones MMTV-specific sequence was present in the amplified shared 96–98 percent homology with numerous strains of 630-bp fragment and that the bands in the agarose gel MMTV and with each other. However, within the highly were specific for MMTV LTR sequences. variable COOH terminus of the sag gene all of the 12 iso- lated clones were either identical or nearly identical to Cloning and sequencing of amplified MMTV-like env from either the MMTV proviral sequence Mtv-8 [20,21] or Mtv- human breast cancer 1 [22,23] as shown in Figure 4. Each of the three family The amplified MMTV env gene-specific 250-bp sequences members contained COOH-terminal sag sequences that present in the primary and metastatic breast tumor tissue were identical or nearly identical (one or two base differ- were cloned using the Invitrogen TOPO TA Cloning kit for ences) to one of these two proviral sequences. Sequencing. A total of 12 clones (4 for each family mem- ber) were sequenced. The sequences of the 12 amplified To analyze relationships between viral strains, phyloge- fragments were shown to be 98.0% – 99.6 percent homol- netic trees have been constructed on the basis of align- ogous to the GR, C3H and BR6 mouse strains of MMTV in ments of LTR ORF sequences [24]. Phylogenetic analysis this 250-bp region of the MMTV env gene. Figure 3 shows of the entire 630 bp LTR ORF sequences isolated from the comparison of the sequences of the 12 clones to the each of the three family members diagnosed with breast three strains of MMTV [17-19] and to each other in this cancer is shown in Figure 5. All 12 of the human MMTV- region of the MMTV env gene. like LTR ORF sag clones segregated to two branches of the MMTV phylogenetic tree, Mtv-8 [20,21] and Mtv-1 As shown in Figure 3 the sequenced env clones fell into 7 [22,23], and did not form a branch of their own. Moreo- classes that differed from one another. However no one ver, clones isolated from the same family member segre- env clone differed from any other by more than 4 base gated to these two separate branches. Interestingly, all of changes either within the same family member or the prior MMTV-like LTR isolates from breast cancer between family members. One env clone each from the [6,16,25], non-Hodgkin's lymphoma [5], and primary mother (M1), father (F2), and daughter (D2) were identi- biliary cirrhosis [26,27] patients also associated with these cal to each other. Two env clones from the father (F3, F4) two branches of the MMTV phylogenetic tree. were identical to each other, one daughter env clone (D1) and one father env clone (F1) were the same, and two env Discussion clones from the mother (M2, M3) were the same. Each To our knowledge, this is the first report of breast cancer family member however, mother (M1, M4), father (F2, in father, mother, and daughter. We acknowledge how- F3, F4), and daughter (D2, D3), each contained env clones ever limitations of our study due to the quality of the DNA that included a single base change mutation denoted by of the archival formalin fixed paraffin samples on which the arrow in Figure 3. Each family member also contained this study is based. Limitations include our inability to env clones (M2, M3, F1, D1, D4) without this single base determine the mutation status of the BRCA1 and BRCA2 Page 4 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 GR/C3H TACATCTGCC TGTGTTACTT ACCCTTATGC CATATTATTA GGATTACCTC BR/6 ------------------- ------------------ ------------------- ------------------- ------------------- ALL 12 -------------------- ------------------ -------------------- -------------------- ------------------- GR/C3H AGCTAATAGA TATAGAGAAA AGAGGATCTA CTTTTCATAT TTCCTGTTCT BR/6 -----T-------------- --------------------- -------------------- ------------------- ------------------- 9 of 12 --------------------- --------------------- -------------------- ------------------- ------------------- F3,F4 -------------------- --------------------- -------------------- ------------------- -------T----------- D3 -----T-------------- --------------------- -------------------- ------------------- -------------------- 2 ↓↓↓↓ GR/C3H TCTTGTAGAT TGACTAATTG TTTAGATTCT TCTGCCTACG ACTATGCAGC BR/6 ------------------- ------------------- -----------C------ ------------------- --------------------- F3,F4 ------------------- ------------------- ------------------- ------------------- -----------A-------- D3 C------------------ C------------------ -------------------- ------------------- -----------A-------- M1,D2.F2 ------------------- ------------------- -------------------- ------------------- -----------A-------- M4 ------------------- ------------------- -------------------- ------------------- -----------A-------- D1,F1 ------------------- -------------------- -------------------- ------------------- ---------------------- M3,M2 -------------------- --------------------- --------------------- ------------------- ---------------------- D4 ------------------- --------------------- ------------C----- -------------------- ---------------------- GR/C3H GATCATAGTC AAGAGGCCGC CATACGTGCT GCTACCTGTA GATATTGGTG BR/6 -------------------- -------------------- --------T---------- ------------------- ------------------- F3,F4 --------------------- -------------------- -------------------- ------------------- -------------------- D3 --G----------------- -------------------- --------------------- ------------------- -------------------- M1,D2, F2-------------------- -------------------- --------------------- ------------------- -------------------- M4 ------------------- --------------------- ---------T---------- ----------------C-- ------------------- D1,F1 --------------------- --------------------- ---------T---------- -------------------- ------------------- M3,M2 --------------------- --------------------- ---------T---------- ----------------C-- ------------------- D4 ------------------- -------------------- ---------T---------- -------------------- -------------------- GR/C3H ATGAACCATG GTTTGATGAT TCTGCCATTC AAACCTTTAG GTATGCCACA BR/6 --------------------- -------------------- ------------------- -------------------- --------------------- F3,F4 --------------------- -------------------- ------------------- -------------------- ---------------------- D3 --------------------- -------------------- -------------------- ------------------- ---------------------- M1,D2,F2--------------------- -------------------- -------------------- -------------------- ---------------------- M4 ---------------------- -------------------- -----C-------------- -------------------- --------------------- D1,F1 ---------------------- -------------------- --------------------- -------------------- ---------------------- M3,M2 ---------------------- -------------------- ----C-------------- -------------------- --------------------- D4 ---------------------- -------------------- -------------------- ------------------- --------------------- a ge Figure 3 Seq nne d daugh u of G ences of R ter , C3 and the 250 H a meta nd B bp PCR MMTV-like sta R6 s tic tbreast rains o tumor t f MMTenv Vissue in lymph gene producnode t ampl of father ified from compared with the DNA of pth rimary breast cancer e sequences in this r tissue of egion of the mother env Sequences of the 250 bp PCR MMTV-like env gene product amplified from the DNA of primary breast cancer tissue of mother and daughter and metastatic breast tumor tissue in lymph node of father compared with the sequences in this region of the env gene of GR, C3H and BR6 strains of MMTV. The numbers 1, 2 and 3 indicate the three locations where the BR6 strain differs from the GR and C3H strains in this region of the MMTV env gene [17,18,19]. The A and ↓ indicate the location at which the identical single base change described in the text occurs. The numbers 7656 and 7905 indicate the location of the MMTV 250 bp env gene sequence within the MMTV genome [17]. Clones are designated as M (mother), D (daughter), and F (father) followed by a number (1–4) denoting the order in which they were cloned. - denotes the same nucleotide. Page 5 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 a Seq m Figure 4 no d Mtv ther a uence h -8 [20,21] to the amp nd da omology of the highly vari ughter and meta lified highly var static br abl ea e CO st tum iaOH ble MMTV or tissue in -terminal sag regio lymp sequences clon n h node of the father of the MMTV ed from sag the primar gene of MMTV provir y breast tuuses Mtv-1 [22,23] mor tissue of the Sequence homology of the highly variable COOH-terminal region of the MMTV sag gene of MMTV proviruses Mtv-1 [22,23] and Mtv-8 [20,21] to the amplified highly variable MMTV sag sequences cloned from the primary breast tumor tissue of the mother and daughter and metastatic breast tumor tissue in lymph node of the father. Clones are named as M (mother), D (daughter), and F (father) followed by a dash and the letter s for sag and numbers (1–4) identifying the order in which they were cloned. - denotes the same nucleotide. Page 6 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 P cluster as Figure 5 hylogeneti two di c analsti ysn isct s of human br pecies east tumor MMTV-like LTR sequences showing that the human and mouse sequences do not Phylogenetic analysis of human breast tumor MMTV-like LTR sequences showing that the human and mouse sequences do not cluster as two distinct species. The 12 human MMTV-like LTR sequences from the three family mem- bers as well as the human sequences previously isolated from human breast tumors, non-Hodgkin's lymphomas, and primary biliary cirrhosis tissue, clustered with their murine counterparts. Boxes denote LTR sequences from mother (M-S1-4), daughter (D-S1-4), and father (F-S1-4). Previously published human isolates AF346815, AY325271, AF243039, AY652977, AY652968, AY652964, AY652975, AY652974, AY652967, AY652969, AY652973, from human breast tumors [6,16,27], AY652970, AY652976, AY652978, AY652965, AY652971, AY652966, AY652972 from human non-Hodgkin's lymphomas [6], and AF513913, AF513923, from human primary biliary cirrhosis patients [26,27]. The mouse sequences, JYG, FM, and SW21 from Asian mice that were used to root the tree, the endogenous MMTV proviral sequences Mtv-8, Mtv-1, and Mtv-6, and the exog- enous MMTV sequences BR6, HEJ, and C3H are bolded. Numbers on branches indicate percent frequencies of assortment in an individual branch after the bootstrap procedure (45) and indicate the robustness of branch assignments. Branch lengths are indicative of the number of nucleotide changes to individual branch points (see scale bar). Page 7 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 genes in these patients [28]. However the late age at which The 630 bp MMTV-like DNA that we have amplified using these breast tumors developed argues against their being primers specific for regions of the ORF of the MMTV LTR caused by mutations in the BRCA1 or BRCA2 genes. codes for the superantigen (sag) gene of MMTV [14,15]. Quantity and quality of the archival DNA also limited us Although highly conserved, the MMTV sag sequences are in that we were able to successfully amplify only parts of not identical with approximately 35 percent of the total two regions, the env and LTR, of the MMTV-like DNA variation clustered at the hypervariable COOH terminus. genome and were not able to detect the presence of inte- This variation present at the COOH terminus of the grated MMTV-like viral sequences in the cellular genome. MMTV sag gene is specific to each MMTV strain. Each of our cloned sag sequences from each family member was Breast cancer in husband and wife may not be as uncom- identical or nearly identical to either the MMTV Mtv-8 mon as generally thought. At least ten couples have been [20,21] or Mtv-1 [22,23] proviral sequence. In a previous previously reported since 1975 [29-33]. Russ and Scanlon publication [6] we have shown that the cloned sag [31] reporting on eleven married couples having histolog- sequences present in both the breast tumor tissue and ically identical neoplasms, including three couples with non-Hodgkin's lymphoma tissue of eight patients diag- breast cancer, noted that the clinical course of the disease nosed with both malignancies also contained sag tended to be similar in both husband and wife, and that sequences that were identical or nearly identical to MMTV husband and wife were diagnosed usually within approx- proviral sequences Mtv-1 and Mtv-8. Similar sequences imately five years of each other. Both observations apply isolated from human breast tumors by others also con- to our family. Some studies however have not shown an tained sag sequences with identity to the Mtv-1 and Mtv-8 increased incidence of breast cancer in wives of men with proviral sequences [16,25]. Also, MMTV-like sag that neoplasm [33-35]. However, Russ and Scanlon noted sequences isolated from patients diagnosed with primary that the tumors they observed in husband and wife have biliary cirrhosis (PBC) also contained sag sequences with been suggested either experimentally or indirectly to have identity to the Mtv-1 and Mtv-8 proviral sequences viral relationships [31]. Lynch et al [36] suggested more [26,27]. Phylogenetic studies from our laboratory and than twenty years ago that a communicable agent might that of others [27] have shown that such sequences segre- play a role in the clustering of certain cancers in spouses. gate to specific branches of the MMTV phylogenetic tree and do not form a distinct branch of their own thus argu- The MMTV-like env sequences that we have detected in the ing for their being mouse derived and not human homo- mother, father, and daughter of this family are 98–99.6 logues of the mouse sequence [6,27]. This study, our percent identical to the GR, C3H and BR6 mouse strains previously published work [6], and that of others [26,27] of MMTV [17-19]. Recently the Env protein of MMTV also indicate that more than one viral strain of MMTV-like from the mouse has been shown to be capable of trans- DNA sequences may be present in the same individual. forming mouse and human mammary epithelial cells in vitro [37]. The MMTV Env protein contains an immunore- Other works that have shown that MMTV-like DNA ceptor tyrosine-based activation motif (ITAM) sequence sequences are for the most part not found in normal cells that appears to allow for its transformation ability [37]. [1-7,25] suggest that the presence of the virus in humans The MMTV-like env gene sequences that we have isolated may result from an exogenous infection. Very recently, a from the primary human breast tumor tissue and the met- virus closely related to the xenotropic murine leukemia astatic breast tumor tissue present in a lymph node in the viruses (MuLVs) has been detected in the tumor tissue of three family members studied in this report and in addi- a certain population of prostate cancer patients. The viral tional breast tumors previously reported and in non- sequence is not found in human genomic DNA thus indi- Hodgkin's lymphomas contain this ITAM sequence [1,6]. cating, as the authors discuss, an exogenous infection It is not yet known if the MMTV-like Env protein coded for [38,39]. The suggestion that MMTV exogenous infection by the MMTV-like DNA sequences which we and others can occur in humans is a highly controversial topic but is have detected in human breast tumors is capable of trans- becoming more plausible with two recent publications by formation. We have sequenced 4 env clones per family Indik et al [40,41]. In these papers the authors show that member for a total of 12 env clones. Seven of these 12 MMTV can infect human cells in vitro, make new MMTV, clones contain an identical single base substitution (Fig- and that this new MMTV can go on to infect other human ure 3, ↓) that we have detected in a number of additional cells. That the MMTV-like DNA sequences that we and breast tumors and in all the non-Hodgkin's lymphomas others have found in human breast tumors may be mouse we have analyzed. This base change of a G to A results in derived is also suggested by the findings that their sag the replacement of an alanine (GCA) with a threonine sequences segregate to arms of the MMTV phylogenetic (ACA) in these samples. Curiously, this single-base tree and do not form a separate branch of their own change occurs within the ITAM domain [37]. [6,26,27]. Additional epidemiological findings suggesting an exogenous infection from mice include the findings of Page 8 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 Stewart et al [42] in which it was shown that the incidence PCR of breast cancer was highest in countries in which the Conditions and primer sequences for MMTV env and LTR mouse strain mus domesticus resides, a mouse strain that gene amplification were those described by Wang et al [4] carries a large number of endogenous MMTV proviruses. and Liu et al [25] respectively. A reaction without template Curiously, a recent study from the Johns Hopkins Univer- DNA was routinely tested to detect possible contamina- sity Schools of Medicine and Public Health has reported tion of master mix components. Reactions with normal that airborne mouse allergen was found in 84 percent of breast DNA were done to rule out contamination of bedrooms of inner city homes in Baltimore and that the tumor samples. The PCR product was analyzed by electro- concentration of this mouse allergen may be similar to phoresis in 1.8 % agarose gels. ΦX174 RF DNA cut with those found in animal facilities [43]. We do not know the the restriction enzyme HaeIII was used as a marker to concentration of mouse allergen in the home of the family identify the size of the PCR products. in this study who lived in a wealthy Baltimore neighbor- hood. Hybridization PCR products were hybridized on Southern blots [13] Conclusion under stringent hybridization conditions to either a 23- The presence of MMTV-like DNA sequences in the malig- base pair (bp) probe specific for DNA sequences present nant tissues of all three family members who lived in the in exogenous MMTV env sequences but not present in same house for decades argues for the possibility of human endogenous retroviral sequence (HERV-K) DNA MMTV as a common environmental etiological agent. [1,4,11,12] or to a 20-bp probe specific for the MMTV LTR Phylogenetic data suggest that the MMTV-like sequences [6,16] and also not present in HERV-K DNA [11,12]. The are mouse and not human derived and that the ultimate 23-bp env probe, which extended from position 7822– reservoir of MMTV is most likely the mouse. Although the 7845 in the MMTV genome, and the 20-bp LTR probe, route by which these three family members came to be which extended from position 972–991 or 9545–9564 in infected with MMTV is unknown, the possibility exists the MMTV genome [17], were end-labeled with [ P]ATP that such infection may have resulted from an exposure to using the T4 kinase forward reaction (Invitrogen Life mice. Technologies, Inc., Carlsbad, CA). Stringent hybridization conditions were as described previously [1,11]. Methods Human tissue Cloning and sequencing of PCR products Formalin-fixed, paraffin-embedded tissue sample blocks Amplified DNA products were cloned directly from the of breast tumor tissue (mother and daughter) and meta- PCR tube using the TOPO TA Cloning kit for Sequencing static breast tumor in lymph node (father) were obtained (Invitrogen). DNA sequencing was performed by Gen- from the Johns Hopkins Medical Center, Baltimore, Mar- emed Synthesis (San Antonio, TX). The resulting yland, courtesy of Drs. Elizabeth Montgomery and Arlene sequences were compared to known published sequences Forastiere, under a protocol approved by the Institutional and to sequences in the Genbank. Review Board of Our Lady of Mercy Medical Center (OLMMC). Hematoxylin and eosin stained slides of each Phylogenetic analysis paraffin block were made and read to determine the loca- The LTR sequences obtained from cloned fragments after PCR amplification were compared to known exogenous tion of malignancy in each block. Blocks were shaved into 5 µm thick serial sections and two sections from each and endogenous MMTV sequences in the Genbank data- specimen were used for DNA extraction. DNA was iso- base. LTR sequences were aligned using DNAssist 2.0 soft- lated from the blocks using a microwave technique [44]. ware and analyzed using the phylogenetic Analysis Using Normal breast tissue samples were obtained from the Parsimony (PAUP 4.0b 10) program [45] as previously Pathology Department at OLMMC under a protocol described [6]. approved by the Institutional Review Board of OLMMC. DNA was extracted from normal breast tissue using the Authors' contributions QIAgen DNA Mini Kit (Qiagen Inc, Germantown, MD). PRE participated in the design of the study, carried out the To determine the quality of the isolated DNA from both molecular biology studies, and drafted the manuscript, paraffin blocks of tumor tissue and fresh normal breast AFRS carried out the phylogenetic studies and participated tissue, globin primers were used in PCR and the resulting in the drafting of the manuscript, PHW participated in the amplified products were electrophoresed in 1.8 % agarose design of the study and in the drafting of the manuscript. gels. Acknowledgements We thank Dr. Stanley Oiseth of the Department of Pathology at Our Lady of Mercy Medical Center (OLMMC) for supplying normal breast tissue and for his expertise in evaluating our paraffin blocks for study and for photos Page 9 of 11 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:2 http://www.infectagentscancer.com/content/3/1/2 of our tissue samples. We thank Drs. Elizabeth Montgomery and Arlene 20. 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