Gefitinib and erlotinib are two new treatments for advanced lung cancer. Gene mutations in the cancer may help predict which patients will respond to these treatments

Lung cancer is the leading cause of death in many countries. To date, cause of cancer death in chemotherapy with cytotoxic agents has been the mainstay of treatment for advanced lung cancer. However, the activity of these agents is quite limited, and they have severe adverse effects. Recent, rapid advances in molecular biology have led to the development of many new agents that inhibit the activities of specific molecules related to tumor growth, invasion, or metastasis  [1] , and these agents have the potential to improve the outcome of lung cancer treatment. But we have not yet managed to successfully deliver these agents from the bench to the bedside.Molecular Therapy for Lung Cancer Gefitinib is the first “molecular-target agent” for lung cancer that inhibits the tyrosine kinase of the epidermal growth factor receptor (EGFR; also known as ERBB1). EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC), especially in squamous cell carcinoma, and its expression is related to the cancer's proliferation. Initial clinical trials of gefitinib showed its modest clinical activity in patients who had failed previous standard chemotherapy. But subsequent randomized trials in patients with previously untreated, advanced NSCLC have not shown a clinical advantage of gefitinib combined with standard chemotherapy over chemotherapy alone [ 2 , 3 , 4 , 5 ]. Erlotinib, another EGFR tyrosine kinase inhibitor for treating NSCLC, which was approved by the United States Food and Drug Administration in November 2004, has a therapeutic profile similar to that of gefitinib. Erlotinib has shown a survival benefit in a phase III trial for chemo-refractory NSCLC that compared erlotinib to best supportive care  [6] . (To date, there have been no trials showing a survival benefit of EGFR inhibitors over standard chemotherapy.) Interestingly, in these trials of EGFR inhibitors, EGFR expression levels in the tumors were not correlated with the response; high response rates were seen in women, patients with adenocarcinoma, nonsmokers, and Japanese patients. Recent studies have now shed light on why certain patients are more likely to respond than others—the key lies in the presence of gene mutations.
Molecular Therapy for Lung Cancer
Gefitinib is the first “molecular-target agent” for lung cancer that inhibits the tyrosine kinase of the epidermal growth factor receptor (EGFR; also known as ERBB1). EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC), especially in squamous cell carcinoma, and its expression is related to the cancer's proliferation. Initial clinical trials of gefitinib showed its modest clinical activity in patients who had failed previous standard chemotherapy. But subsequent randomized trials in patients with previously untreated, advanced NSCLC have not shown a clinical advantage of gefitinib combined with standard chemotherapy over chemotherapy alone [ 2 , 3 , 4 , 5 ].Erlotinib, another EGFR tyrosine kinase inhibitor for treating NSCLC, which was approved by the United States Food and Drug Administration in November 2004, has a therapeutic profile similar to that of gefitinib. Erlotinib has shown a survival benefit in a phase III trial for chemo-refractory NSCLC that compared erlotinib to best supportive care  [6] . (To date, there have been no trials showing a survival benefit of EGFR inhibitors over standard chemotherapy.) Interestingly, in these trials of EGFR inhibitors, EGFR expression levels in the tumors were not correlated with the response; high response rates were seen in women, patients with adenocarcinoma, nonsmokers, and Japanese patients. Recent studies have now shed light on why certain patients are more likely to respond than others—the key lies in the presence of gene mutations.EGFR Gene Mutations in NSCLC In April and May 2004, two new studies published in  Science  and the  New England Journal of Medicine  helped to explain at the molecular level the low clinical activity of EGFR inhibitors [ 7 , 8 ]. These studies identified somatic mutations in the EGFR gene, especially around the region encoding the ATP-binding pocket of the receptor's tyrosine kinase domain. These mutations increased the sensitivity of tumor cells to gefitinib. A high incidence of mutations was detected in patients with NSCLC that had a durable clinical response to gefitinib, and subsequent studies revealed that these mutations were also related to the response to erlotinib. Moreover, such mutations were more frequently detected in patients with adenocarcinoma, in women, in Japanese patients, and in nonsmokers  [9] —results that were compatible with previous clinical data. These findings should hopefully lead to the identification of subgroups of patients who are likely to benefit substantially from such EGFR inhibitors. But another question was raised by these studies: is it only EGFR gene mutations that determine the response to EGFR inhibitors?
EGFR Gene Mutations in NSCLC
In April and May 2004, two new studies published in  Science  and the  New England Journal of Medicine  helped to explain at the molecular level the low clinical activity of EGFR inhibitors [ 7 , 8 ]. These studies identified somatic mutations in the EGFR gene, especially around the region encoding the ATP-binding pocket of the receptor's tyrosine kinase domain. These mutations increased the sensitivity of tumor cells to gefitinib. A high incidence of mutations was detected in patients with NSCLC that had a durable clinical response to gefitinib, and subsequent studies revealed that these mutations were also related to the response to erlotinib. Moreover, such mutations were more frequently detected in patients with adenocarcinoma, in women, in Japanese patients, and in nonsmokers  [9] —results that were compatible with previous clinical data.ScienceNew England Journal of MedicineThese findings should hopefully lead to the identification of subgroups of patients who are likely to benefit substantially from such EGFR inhibitors. But another question was raised by these studies: is it only EGFR gene mutations that determine the response to EGFR inhibitors?RAS Gene Mutations in NSCLC The RAS proteins are low-molecular-weight GTPases that are bound to the inner side of the cell membrane. They are involved in signal transduction pathways: they regulate downstream effector proteins such as Raf/MAP kinase and PI3 kinase, under the influence of various cell surface receptors including EGFR ( Figure 1 ). Mutations of the K-ras gene have been found in up to 30% of lung adenocarcinomas and have been considered a poor prognostic factor  [10] . A study by Pao and colleagues published in this issue of  PLoS Medicine  suggests that the K-ras mutation is an important predictive factor in defining which patients will benefit from receiving EGFR inhibitors  [11] . In Pao and colleagues' study, the K-ras mutations were completely associated with a lack of response to EGFR inhibitors (0/14 tumors with K-ras mutations were sensitive). The EGFR mutations were significantly related to response (17/17 tumors with EGFR mutations were sensitive), as observed in previous studies. Figure 1 EGFR Signal Transduction in Cancer Cells Arrows indicate stimulation, and T-bars, inhibition. EGFR-I, EGFR inhibitor; MEK, MAPK kinase. Although the number of tumors examined in this study may be too small to lead to a definite conclusion—and, furthermore, about half of tumors were retrospectively collected—this study is the first to show that mutations of EGFR and K-ras are not related and that K-ras mutations are associated with a lack of sensitivity to EGFR inhibitors. However, since the sensitivity of the method for finding each mutation influences how often it is detected, standardization of detection methods is important. Hence, the true incidence of K-ras mutations in NSCLC (including non-adenocarcinoma) and their refractoriness to EGFR inhibitors need to be established in further studies. In fact, the reported incidence of EGFR mutations differs between institutions. In addition, although Pao and colleagues' study examined such mutations only by DNA sequence, mRNA or protein expression would show mutation status more accurately.
RAS Gene Mutations in NSCLC
The RAS proteins are low-molecular-weight GTPases that are bound to the inner side of the cell membrane. They are involved in signal transduction pathways: they regulate downstream effector proteins such as Raf/MAP kinase and PI3 kinase, under the influence of various cell surface receptors including EGFR ( Figure 1 ). Mutations of the K-ras gene have been found in up to 30% of lung adenocarcinomas and have been considered a poor prognostic factor  [10] . A study by Pao and colleagues published in this issue of  PLoS Medicine  suggests that the K-ras mutation is an important predictive factor in defining which patients will benefit from receiving EGFR inhibitors  [11] . In Pao and colleagues' study, the K-ras mutations were completely associated with a lack of response to EGFR inhibitors (0/14 tumors with K-ras mutations were sensitive). The EGFR mutations were significantly related to response (17/17 tumors with EGFR mutations were sensitive), as observed in previous studies.PLoS MedicineAlthough the number of tumors examined in this study may be too small to lead to a definite conclusion—and, furthermore, about half of tumors were retrospectively collected—this study is the first to show that mutations of EGFR and K-ras are not related and that K-ras mutations are associated with a lack of sensitivity to EGFR inhibitors. However, since the sensitivity of the method for finding each mutation influences how often it is detected, standardization of detection methods is important. Hence, the true incidence of K-ras mutations in NSCLC (including non-adenocarcinoma) and their refractoriness to EGFR inhibitors need to be established in further studies. In fact, the reported incidence of EGFR mutations differs between institutions. In addition, although Pao and colleagues' study examined such mutations only by DNA sequence, mRNA or protein expression would show mutation status more accurately.Therapeutic Implications In the clinical setting, prolonged disease stabilization with no measurable reduction in tumor size is seen in about half of patients treated with EGFR inhibitors, and a significant survival benefit in this group was shown in a phase III trial  [6] . The mutation status of the EGFR gene and K-ras gene among those stabilized tumors should be evaluated; it may reveal two different groups differentiated by mutation status. It will also be important to establish whether, in tumors with an EGFR mutation that eventually acquire resistance to EGFR inhibitors, the resistance is associated with a change of EGFR status (mutation or change in expression) or is associated with a change of K-ras status. Such changes can be revealed by re-examination of tumors at the time of relapse. If resistance is K-ras dependent, the new “K-ras inhibitors” (unfortunately not yet available) may be of help for patients who have developed a K-ras mutation, as well as for patients whose tumors harbor K-ras mutations from the beginning. Although many issues still need to be resolved step by step through prospective trials to show the benefit of this new strategy, these mutations are promising predictive factors for the success of EGFR inhibitors. Even if the population who may benefit from EGFR inhibitors (such as patients who are positive for an EGFR mutation and negative for a K-ras mutation) is very small, the response rate of over 80% is encouraging, and has never before been achieved in advanced NSCLC. Finally, it is important to detect which patients derive no benefit from EGFR inhibitors because severe adverse effects such as acute lung injury can occur in any patient treated with these drugs  [12] . By combining all the factors that relate to response or resistance, patients who will benefit from treatment can hopefully be identified. Undoubtedly we have taken a great step forward in molecular therapy for lung cancer treatment.
Therapeutic Implications
In the clinical setting, prolonged disease stabilization with no measurable reduction in tumor size is seen in about half of patients treated with EGFR inhibitors, and a significant survival benefit in this group was shown in a phase III trial  [6] . The mutation status of the EGFR gene and K-ras gene among those stabilized tumors should be evaluated; it may reveal two different groups differentiated by mutation status.It will also be important to establish whether, in tumors with an EGFR mutation that eventually acquire resistance to EGFR inhibitors, the resistance is associated with a change of EGFR status (mutation or change in expression) or is associated with a change of K-ras status. Such changes can be revealed by re-examination of tumors at the time of relapse. If resistance is K-ras dependent, the new “K-ras inhibitors” (unfortunately not yet available) may be of help for patients who have developed a K-ras mutation, as well as for patients whose tumors harbor K-ras mutations from the beginning.Although many issues still need to be resolved step by step through prospective trials to show the benefit of this new strategy, these mutations are promising predictive factors for the success of EGFR inhibitors. Even if the population who may benefit from EGFR inhibitors (such as patients who are positive for an EGFR mutation and negative for a K-ras mutation) is very small, the response rate of over 80% is encouraging, and has never before been achieved in advanced NSCLC. Finally, it is important to detect which patients derive no benefit from EGFR inhibitors because severe adverse effects such as acute lung injury can occur in any patient treated with these drugs  [12] . By combining all the factors that relate to response or resistance, patients who will benefit from treatment can hopefully be identified. Undoubtedly we have taken a great step forward in molecular therapy for lung cancer treatment.


Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.

Findings Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that is the leading cause of congenital viral infection in the United States and Europe. Intrauterine transmission of the virus occurs in approximately 40% of pregnant women with primary HCMV infection, and the incidence of congenital HCMV infection is an estimated 1% of newborns [ 1 - 3 ]. Although the pathogenesis of HCMV transmission to the fetus during pregnancy is unclear, the placenta has been implicated as an important determining factor [ 4 - 8 ]. Primary first trimester extravillous cytotrophoblasts (CTBs), which are specialized placental epithelial cells that invade and remodel the uterine wall during placentation, have been previously shown to be fully permissive for HCMV infection  in vitro  [ 7 , 9 ]. Additionally, using an  in vitro  coculture system, Maidji and colleagues demonstrated that infected uterine microvascular endothelial cells transmit HCMV to differentiating invading CTBs, suggesting that placental HCMV infection can occur in a retrograde fashion that initiates in the maternal endothelium [ 8 ]. Despite these reports, the minimal mitotic potential of primary CTBs restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. Alternatively, the utilization of trophoblast cell lines would provide an easily manipulative  in vitro  model for the study of HCMV infection of the placenta. In the present study, we used a first trimester human extravillous CTB cell line, termed SGHPL-4, to investigate HCMV replication. SGHPL-4 cells were derived from first trimester chorionic villous tissue and have been described previously. Importantly, these cells share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotrophin, and human placental lactogen[ 10 - 12 ]. The lytic replication cycle of HCMV is a temporally regulated cascade of events that is initiated when the virus binds to host cell receptors. Upon entry into the cell, the viral DNA translocates to the nucleus where viral gene expression occurs in a stepwise fashion beginning with the expression of immediate early (IE) genes (reviewed in [ 13 ]). To initiate studies of HCMV infection in the SGHPL-4 cell line, placental CTBs and human foreskin fibroblasts (HFFs) were infected with HCMV and the nuclear HCMV IE proteins (IE 1/2; Chemicon, Temecula, CA) were examined by immunofluorescence at various intervals after viral infection. At 3 h p.i., IE 1/2 was present in SGHPL-4 cells in similar numbers to that of HFFs. In fact, the percentages of IE-positive cells initially did not differ between CTBs and HFFs, suggesting that viral entry into the cells and IE transcription occurred at similar rates between the cell types (Figure  1A,1B,1C ). Characteristic cytopathic effects of HCMV infection including swollen cells with nuclear inclusions were observed in both SGHPL-4 and HFF cells by 48 h p.i. (data not shown), and throughout a 6 day culture period, the numbers of IE-positive cells increased continuously in both cell types (Figure  1A ). Interestingly, the rate of IE 1/2 protein expression in SGHPL-4 cells as compared to HFFs appeared to differ beginning at 72 h p.i. By 72 h p.i., there was a 40% increase in the percentage of IE-positive HFFs over SGHPL-4 cells. While nearly 100% of HFFs stained positive for IE 1/2 120 h (5 days) p.i., the maximum fraction of IE-positive SGHPL-4 cells did not exceed 60% (Figure  1A,1D,1E ), suggesting that subsequent viral gene expression and thus cell-to-cell viral spread may be kinetically delayed. These findings are consistent with other reports demonstrating delayed kinetics of viral gene expression in primary CTBs as compared to primary fibroblasts [ 14 ]. Figure 1 Productive HCMV infection in SGHPL-4 and HFF cells.  (A-E) HCMV IE protein expression in human cytotrophoblasts. SGHPL-4 (□) or HFF (■) cells were infected with HCMV strain RVdlMwt-GFP [17] at a MOI of 2.5 PFU per cell and incubated at 37°C for 1, 4, 8, 12, 24, 48, 72, 96,120 or 144 h. At the indicated times, cells were fixed and stained for HCMV IE 1/2 and DAPI (Molecular Probes) and visualized on a Zeiss Axio Plan II microscope (Thornwood, NY). To determine the number of HCMV-infected cells, three fields of view were considered and the percent of IE-positive cells was calculated as: (average number of IE-stained cells/average number of DAPI-stained cells) × 100. The graph demonstrates an increase in the percentage of SGHPL-4 and HFF cells expressing IE 1/2 over a period of time. Representative images of HCMV IE 1/2 are depicted at 8 h p.i in (B) CTBs and (C) HFFs and at 120 h p.i. in (D) CTBs and (E) HFFs; IE 1/2-red, DAPI-blue, overlaid-purple. (F) Infected CTBs produce and release infectious virions. SGHPL-4 or HFF cells were inoculated with HCMV at a MOI of 0.1 PFU per cell. At the indicated times, cells or culture medium were harvested, freeze-thawed three times, and titers of infectious virus in SGHPL-4 cell lysates (○) and supernatants (△) and HFF cell lysates (●) and supernatants (▲) were determined by a microtiter plaque assay on HFFs [18]. Infectious progeny virus was detected in both cell lysates and culture supernatants of SGHPL-4 and HFF cells. The dashed line represents the lower limit of detection of the plaque assay used to measure viral titers. Although several studies have shown that first trimester primary trophoblasts can be permissively infected with HCMV, some reports have demonstrated that progression through the replicative cycle was slow and progeny virus remained predominantly cell associated [ 9 , 15 , 16 ]. To determine whether SGHPL-4 cells support productive HCMV replication, 9 day viral growth assays were performed (Figure  1F ). SGHPL-4 and HFF cells were inoculated with HCMV at a MOI of 0.1 PFU per cell, and both culture lysates and supernatants were titered for infectious virus at various days p.i. While viral titers in infected HFFs were detectable as early as 2 days p.i., viral replication was undetectable or below the lower limit of detection of the assay in SGHPL-4 lysates up to 3 days p.i. However, at days 5–9 p.i., HCMV replicated to titers of ≥ 5000 and 3600 PFU/ml in SGHPL-4 cell lysates and supernatants, respectively. Relative to HFF-infected control cultures, viral titers recovered from SGHPL-4 culture lysates and supernatants were reduced by ~20- and ~200-fold, respectively (Figure  1F ). While viral titers were decreased in infected SGHPL-4 cells as compared to infected HFFs, placental CTBs effectively supported productive viral replication as measured by infectious intracellular and extracellular virions. Moreover, when SGHPL-4 cells were infected with another laboratory-derived strain of HCMV (strain AD169), similar results were obtained (data not shown) suggesting that viral replication was not virus-strain specific. Collectively, these data indicate that SGHPL-4 cells support productive HCMV replication. In the present study, we demonstrate that the first trimester extravillous CTB cell line SGHPL-4 is fully permissive for HCMV replication. The utilization of a CTB cell line, rather than primary CTBs and explant cultures that are short-lived cultures, may provide an experimental advantage for  in vitro  studies of placental HCMV infection. We propose that the permissiveness for HCMV replication in SGHPL-4 cells may allow for future studies in elucidating the molecular mechanism(s) of HCMV infection and pathogenesis at the maternal-fetal interface during early pregnancy.
Findings
Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that is the leading cause of congenital viral infection in the United States and Europe. Intrauterine transmission of the virus occurs in approximately 40% of pregnant women with primary HCMV infection, and the incidence of congenital HCMV infection is an estimated 1% of newborns [ 1 - 3 ]. Although the pathogenesis of HCMV transmission to the fetus during pregnancy is unclear, the placenta has been implicated as an important determining factor [ 4 - 8 ]. Primary first trimester extravillous cytotrophoblasts (CTBs), which are specialized placental epithelial cells that invade and remodel the uterine wall during placentation, have been previously shown to be fully permissive for HCMV infection  in vitro  [ 7 , 9 ]. Additionally, using an  in vitro  coculture system, Maidji and colleagues demonstrated that infected uterine microvascular endothelial cells transmit HCMV to differentiating invading CTBs, suggesting that placental HCMV infection can occur in a retrograde fashion that initiates in the maternal endothelium [ 8 ]. Despite these reports, the minimal mitotic potential of primary CTBs restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. Alternatively, the utilization of trophoblast cell lines would provide an easily manipulative  in vitro  model for the study of HCMV infection of the placenta. In the present study, we used a first trimester human extravillous CTB cell line, termed SGHPL-4, to investigate HCMV replication. SGHPL-4 cells were derived from first trimester chorionic villous tissue and have been described previously. Importantly, these cells share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotrophin, and human placental lactogen[ 10 - 12 ].in vitroin vitroin vitroThe lytic replication cycle of HCMV is a temporally regulated cascade of events that is initiated when the virus binds to host cell receptors. Upon entry into the cell, the viral DNA translocates to the nucleus where viral gene expression occurs in a stepwise fashion beginning with the expression of immediate early (IE) genes (reviewed in [ 13 ]). To initiate studies of HCMV infection in the SGHPL-4 cell line, placental CTBs and human foreskin fibroblasts (HFFs) were infected with HCMV and the nuclear HCMV IE proteins (IE 1/2; Chemicon, Temecula, CA) were examined by immunofluorescence at various intervals after viral infection. At 3 h p.i., IE 1/2 was present in SGHPL-4 cells in similar numbers to that of HFFs. In fact, the percentages of IE-positive cells initially did not differ between CTBs and HFFs, suggesting that viral entry into the cells and IE transcription occurred at similar rates between the cell types (Figure  1A,1B,1C ). Characteristic cytopathic effects of HCMV infection including swollen cells with nuclear inclusions were observed in both SGHPL-4 and HFF cells by 48 h p.i. (data not shown), and throughout a 6 day culture period, the numbers of IE-positive cells increased continuously in both cell types (Figure  1A ). Interestingly, the rate of IE 1/2 protein expression in SGHPL-4 cells as compared to HFFs appeared to differ beginning at 72 h p.i. By 72 h p.i., there was a 40% increase in the percentage of IE-positive HFFs over SGHPL-4 cells. While nearly 100% of HFFs stained positive for IE 1/2 120 h (5 days) p.i., the maximum fraction of IE-positive SGHPL-4 cells did not exceed 60% (Figure  1A,1D,1E ), suggesting that subsequent viral gene expression and thus cell-to-cell viral spread may be kinetically delayed. These findings are consistent with other reports demonstrating delayed kinetics of viral gene expression in primary CTBs as compared to primary fibroblasts [ 14 ].Although several studies have shown that first trimester primary trophoblasts can be permissively infected with HCMV, some reports have demonstrated that progression through the replicative cycle was slow and progeny virus remained predominantly cell associated [ 9 , 15 , 16 ]. To determine whether SGHPL-4 cells support productive HCMV replication, 9 day viral growth assays were performed (Figure  1F ). SGHPL-4 and HFF cells were inoculated with HCMV at a MOI of 0.1 PFU per cell, and both culture lysates and supernatants were titered for infectious virus at various days p.i. While viral titers in infected HFFs were detectable as early as 2 days p.i., viral replication was undetectable or below the lower limit of detection of the assay in SGHPL-4 lysates up to 3 days p.i. However, at days 5–9 p.i., HCMV replicated to titers of ≥ 5000 and 3600 PFU/ml in SGHPL-4 cell lysates and supernatants, respectively. Relative to HFF-infected control cultures, viral titers recovered from SGHPL-4 culture lysates and supernatants were reduced by ~20- and ~200-fold, respectively (Figure  1F ). While viral titers were decreased in infected SGHPL-4 cells as compared to infected HFFs, placental CTBs effectively supported productive viral replication as measured by infectious intracellular and extracellular virions. Moreover, when SGHPL-4 cells were infected with another laboratory-derived strain of HCMV (strain AD169), similar results were obtained (data not shown) suggesting that viral replication was not virus-strain specific. Collectively, these data indicate that SGHPL-4 cells support productive HCMV replication.In the present study, we demonstrate that the first trimester extravillous CTB cell line SGHPL-4 is fully permissive for HCMV replication. The utilization of a CTB cell line, rather than primary CTBs and explant cultures that are short-lived cultures, may provide an experimental advantage for  in vitro  studies of placental HCMV infection. We propose that the permissiveness for HCMV replication in SGHPL-4 cells may allow for future studies in elucidating the molecular mechanism(s) of HCMV infection and pathogenesis at the maternal-fetal interface during early pregnancy.in vitroList of abbreviations human cytomegalovirus (HCMV), cytotrophoblast (CTB), human foreskin fibroblasts (HFFs), immediate early (IE), hours (h), post-infection (p.i.), multiplicity of infection (MOI), plaque forming unit (PFU), 4', 6-diamidino-2-phenylindole, dihydrochloride(DAPI)
List of abbreviations
human cytomegalovirus (HCMV), cytotrophoblast (CTB), human foreskin fibroblasts (HFFs), immediate early (IE), hours (h), post-infection (p.i.), multiplicity of infection (MOI), plaque forming unit (PFU), 4', 6-diamidino-2-phenylindole, dihydrochloride(DAPI)Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions HL participated in the experimental design, performed all experiments and drafted the manuscript. BS participated in the experimental design and assisted with viral propagation and viral replication assays. CM conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
Authors' contributions
HL participated in the experimental design, performed all experiments and drafted the manuscript. BS participated in the experimental design and assisted with viral propagation and viral replication assays. CM conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.